Oil degumming methods

ABSTRACT

In alternative embodiments, the invention provides phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes, nucleic acids encoding them, antibodies that bind specifically to them, and methods for making and using them. Industrial methods and products comprising use of these phospholipases are also provided. In certain embodiments, provided herein are methods for hydration of non hydratable phospholipids (NHPs) within a lipid matrix. The methods enable migration of NHPs to an oil-water interface thereby allowing the NHPs to be reacted and/or removed from the lipids. In certain embodiments, provided is a method for removing NHPs, hydratable phospholipids, and lecithins from vegetable oils to produce a degummed oil or fat product that can be used for food production and/or non-food applications. In certain embodiments, provided herein are methods for hydration of NHPs followed by enzymatic treatment and removal of various phospholipids and lecithins. The methods provided herein can be practiced on either crude or water-degummed oils.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.61/252,638 filed Oct. 16, 2009. The disclosure of the above referencedapplication is incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application is a National stage under 35 U.S.C. 371 (c) ofInternational Application No. PCT/US2010/051920, filed Oct. 8, 2010,which claims priority to U.S. provisional application No. 61/252,638filed Oct. 16, 2009. The disclosure of each of the above referencedapplications is incorporated by reference herein in its entirety

File Name Date of Creation Size 011631-0051-888_SeqListing.txt Oct. 15,2009 17.4 kb

FIELD OF THE INVENTION

This invention relates generally to phospholipase enzymes,polynucleotides encoding the enzymes, methods of making and using thesepolynucleotides and polypeptides. In alternative embodiments, theinvention provides phosphatidylinositol-specific phospholipase C(PI-PLC) enzymes, nucleic acids encoding them, antibodies that bindspecifically to them, and methods for making and using them. Industrialmethods and products comprising use of these phospholipases areprovided. Also provided herein are methods for hydration of nonhydratable phospholipids (NHPs) within a lipid matrix. The methodsenable migration of NHPs to an oil-water interface thereby allowing theNHPs to be reacted and/or removed from the lipids. In certainembodiments, provided are methods for removing NHPs, hydratablephospholipids, and lecithins (known collectively as “gums”) fromvegetable oils to produce a degummed oil or fat product that can be usedfor food production and/or non-food applications. In certainembodiments, provided herein are methods for hydration of NHPs followedby enzymatic treatment and removal of various phospholipids andlecithins. The methods provided herein can be practiced on either crudeor water-degummed oils. In certain embodiment, provided herein aremethods for obtaining phospholipids from an edible oil.

BACKGROUND

Crude vegetable oils obtained from either pressing or solvent extractionmethods are a complex mixture of triacylglycerols, phospholipids,sterols, tocopherols, free fatty acids, trace metals, and other minorcompounds. It is desirable to remove the phospholipids, free fatty acidsand trace metals in order to produce a quality salad oil with a blandtaste, light color, and a long shelf life or oil suitable fortransformation into a feedstock ready for chemical or enzymaticconversion into a biofuel (methyl- or ethyl-esters), bio-plastic(epoxidized oil), and other traditional petroleum based materials.

The removal of phospholipids generates almost all of the lossesassociated with the refining of vegetable oils. Most phospholipidmolecules possess both a hydrophilic functional group and lipophilicfatty acid chains, they tend to be excellent natural emulsifiers. Thefunctional group in phospholipids may be any of several of a variety ofknown types, a few of which are illustrated in scheme 1 below.

Phospholipids containing the functional groups -choline, -inositol and-ethanolamine have the greatest affinity for water, while the acids,acid salts (Calcium (Ca), Magnesium (Mg), and Iron (Fe)), and-ethanolamine salts (Ca, Mg, and Fe) have much lower affinities forwater. Phosphatidic acid and the salts of phosphatidic acid are commonlyknown as “non hydratable phospholipids” or NHPs. Table 1 containsrelative rates of hydration of different phospholipids as reported bySen Gupta, A. K., Fette Seifen Anstrichmittel 88 pages 79-86 (1986). andlater by Segers, J. C., et al., “Degumming—Theory and Practice”published by American Oil Chemists's Society in “Edible fats and Oilsprocessing: basic principals and modern practices: World conferenceproceedings”/edited by David Erickson, (1990) pages 88-93.

TABLE 1 Relative Rates of Hydration Phospholipids Relative Rate ofHydration Phosphatidyl Choline (PC) 100 Phosphatidyl Inositol (PI) 44Calcium Salt of Phosphatidyl Inositol 24 Phosphatidyl Ethanolamine (PE)16 Phosphatidic Acid (PA) 8.5 Calcium Salt of Phosphatidyl Ethanolamine0.9 Calcium Salt of Phosphatidic Acid 0.6

Calcium, magnesium, and iron salts of phospholipids are formed by anenzyme present in oilseeds, phospholipase D (PLD). The enzyme remainsdormant within the mature seed until the protective coating of the seedhas been damaged during storage or seed “preparations” prior to removalof the oil. The reaction of PLD within the seed will cleave the-choline, -inositol, -serine or -ethanolamine from the phosphate groupyielding the Phosphatidic Acid (PA). Additionally, since the cleavageoccurs in the presence of an abundance of divalent metals (Ca, Mg, andFe), the NHPs are formed. The phosphatidic acid calcium ion complex isshown below:

Phospholipids are commonly measured in oil as “phosphorus content” inparts per million. Table 2 sets forth the typical amounts ofphospholipids present in the major oilseed crops, and the distributionof the various functional groups as a percentage of the phospholipidspresent in the oil.

TABLE 2 Typical levels and phospholipid distributions for commonoilseeds Soy Oil Canola Oil Sunflower Oil Phosphorus (ppm)  400-1500200-900 300-700 PC (%) 12-46 25-40 29-52 PE (%)  8-34 15-25 17-26 PA (%) 2-21 10-20 15-30 PS (%) <0.5 <0.5 <0.5 PI (%)  2-15  2-25 11-22

Table 3 below provides typical phospholipid amounts and distributionsfor soybean gums. In Table 3, “as is” means the typical phospholipidcomposition removed from vegetable oil with the entrained oil (2molecules of phospholipids and 1 molecule of oil), yielding an AcetoneInsoluble content of 67%. “Normalized” means the phospholipidcomposition without any oil present, yielding an Acetone Insolublecontent of 100%.

TABLE 3 Typical phospholipid amounts and distributions for soybean gumsPercentage Percentage “As-Is” “Normalized” Phosphatidyl Choline (PC)33.9 47.2 Phosphatidyl Ethanolamine (PE) 14.3 19.9 Phosphatidyl Serine(PS) 0.4 0.6 Phosphatidic Acid (PA) 6.4 8.9 Phosphatidyl Inositol (PI)16.8 23.4 Total 71.8 100.0

The conversion of phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and phosphatidic acid intoeither their lyso- or phospho-forms greatly changes the economics of thedegumming in a modern industrial refining operation. The conversion ofall of the phospholipids into their lyso-forms eliminating the neutraloil loss represents an increase in oil yield of up to 1.4%, whileconverting all the phospholipids into their phospho-forms represents anoil yield increase up to 3.0% for a crude oil over water degummingcontaining 1000 ppm of phosphorus.

Phospholipids can be partially or totally removed from vegetable oilsthrough several different known means. The most commonly used processesin the industry are water degumming, acid degumming, caustic refiningand enzymatic degumming Exemplary processes are described in U.S. Pat.Nos. 4,049,686; 4,698,185; 5,239,096; 5,264,367; 5,286,886; 5,532,163;6,001,640; 6,103,505; U.S. Pat. Nos. 6,127,137; 6,143,545; 6,172,248;6,548,633; 7,494,676; and 7,226,771, and U.S. publication nos.2007/0134777, 2005/0059130, 2008/0182322, and 2009/0069587.

The existing methods are not sufficient to remove or reactnon-hydratable phospholipids present in the oil because the NHPs are notavailable to be hydrated or reacted to enable their removal.

There is a need for cost effective and efficient methods for removingNHPs, hydratable phospholipids, and lecithins (known collectively as“gums”) from vegetable oils to produce a degummed oil or fat productthat can be used for food production and/or non-food applications.

Phospholipases are enzymes that hydrolyze the ester bonds ofphospholipids. Corresponding to their importance in the metabolism ofphospholipids, these enzymes are widespread among prokaryotes andeukaryotes. The phospholipases affect the metabolism, construction andreorganization of biological membranes and are involved in signalcascades. Several types of phospholipases are known which differ intheir specificity according to the position of the bond attacked in thephospholipid molecule.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes are afamily of eukaryotic intracellular enzymes that play an important rolein signal transduction processes. The PI-PLC catalyzed reaction is:1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate (also called PIP₂,phosphatidylinositol bisphosphate)+H₂O

1D-myo-inositol 1,4,5-trisphosphate (also called IP₃, inositoltriphosphate)+diacylglycerol

Families of phospholipase C (PLC) enzymes have been identified inbacteria and in eukaryotic trypanosomes. PLC enzymes belong to thefamily of hydrolases and phosphodiesterases. PLC participate inphosphatidylinositol 4,5-bisphosphate (PIP₂) metabolism and lipidsignaling pathways in a calcium-dependent manner. PLC isoforms candiffer in their mode of activation, expression levels, catalyticregulation, cellular localization, membrane binding avidity and tissuedistribution. All are capable of catalyzing the hydrolysis of PIP₂ intotwo important second messenger molecules, which go on to alter cellresponses such as proliferation, differentiation, apoptosis,cytoskeleton remodeling, vesicular trafficking, ion channel conductance,endocrine function and neurotransmission. PLCs are described in, forexample, Carmen, G., J. Biol. Chem. 270 (1995) 18711-18714, Jianag, Y.,J. Biol. Chem., 271 (1996) 29528-29532, Waggoner, D., J. Biol. Chem. 270(1995) 19422-19429, Molecular Probes Product Sheet 2001, and Sano etal., Am. J. Physiol. Lung Cell Mol. Physiol. 281:844-851, 2001.

Phospholipase A1 (PLA1) enzymes remove the 1-position fatty acid toproduce free fatty acid and 1-lyso-2-acylphospholipid. Phospholipase A2(PLA2) enzymes remove the 2-position fatty acid to produce free fattyacid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intra-or extra-cellular, membrane-bound or soluble. Intracellular PLA2 isfound in almost every mammalian cell. Phospholipase C (PLC) enzymesremove the phosphate moiety to produce 1,2 diacylglycerol and aphosphate ester. Phospholipase D (PLD) enzymes produce1,2-diacylglycerophosphate and base group.

SUMMARY OF THE INVENTION

Provided herein are polypeptides and polynucleotides encodingpolypeptides having a phosphatidylinositol-specific phospholipase C(PI-PLC) or equivalent enzyme activity, and/or another phospholipaseactivity, including a phospholipase A, B, C, D, patatin, phosphatidicacid phosphatases (PAP) and/or lipid acyl hydrolase (LAH) or equivalentenzyme activity, and methods of making and using these polynucleotidesand polypeptides. In one aspect, provided herein are polypeptides, e.g.,enzymes, having a phospholipase activity, e.g., phospholipase A, B, D orC activity, e.g. phosphatidylinositol-specific phospholipase C (PI-PLC)activity. The enzymatic activities of the polypeptides and peptides asprovided herein include (comprise or consist of) a phospholipaseactivity, a phospholipase C activity, or a phosphatidylinositol-specificphospholipase C (PI-PLC) activity, including hydrolysis of lipids,acidolysis reactions (e.g., to replace an esterified fatty acid with afree fatty acid), transesterification reactions (e.g., exchange of fattyacids between triacylglycerides), ester synthesis, ester interchangereactions and lipid acyl hydrolase (LAH) activity. In another aspect,the polypeptides as provided herein are used to synthesizeenantiomerically pure chiral products. The polypeptides as providedherein can be used in a variety of pharmaceutical, agricultural andindustrial contexts, including the manufacture of cosmetics andnutraceuticals. Additionally, the polypeptides as provided herein can beused in food processing, brewing, bath additives, alcohol production,peptide synthesis, enantioselectivity, hide preparation in the leatherindustry, waste management and animal waste degradation, silver recoveryin the photographic industry, medical treatment, silk degumming, biofilmdegradation, biomass conversion to ethanol, biodefense, antimicrobialagents and disinfectants, personal care and cosmetics, biotech reagents,in increasing starch yield from corn wet milling, and as pharmaceuticalssuch as digestive aids and anti-inflammatory (antiphlogistic) agents.

In certain embodiments, provided herein are compositions (e.g.,phospholipase, phospholipase C, phosphatidylinositol-specificphospholipase C (PI-PLC)) and methods for producing low phospholipidoils, e.g., oils with a lower phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,and/or phosphatidic acid content. Any oil, e.g. vegetable oil, e.g.canola oil, soybean oil, or animal oil or fat, e.g., tallow, can betreated with a composition, or by a method, as provided herein. Anyfoods, edible items, or baking, frying or cooking products (e.g.,sauces, marinades, condiments, spray oils, margarines, baking oils,mayonnaise, cooking oils, salad oils, spoonable and pourable dressings,and the like, and products made therewith) can comprise a vegetable oilor animal fat that has been treated with a composition or by a method asprovided herein. Vegetable oils modified to be lower phospholipid oilscan be used in any foods, edible items or baking or cooking products,e.g., sauces, marinades, condiments, spray oils, margarines, bakingoils, mayonnaise, cooking oils, salad oils, spoonable and pourabledressings and the like. In one embodiment, provided herein are oils,such as vegetable oils, e.g., canola oil or soybean oil, and foods orbaking or cooking products, including sauces, marinades, condiments,spray oils, margarines, mayonnaise, baking oils, cooking oils, flyingoils, salad oils, spoonable and pourable dressings, and the like,wherein the oil or food, baking or cooking product has been modifiedusing an enzyme as provided herein. In one aspect, these vegetable oils,e.g. canola oil, castor oil, coconut oil, coriander oil, corn oil,cottonseed oil, hazelnut oil, hempseed oil, linseed oil, meadowfoam oil,olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, ricebran oil, safflower oil, sasanqua oil, soybean oil, sunflower seed oil,tall oil, tsubaki oil, varieties of “natural” oils having altered fattyacid compositions via Genetically Modified Organisms (GMO) ortraditional “breeding” such as high oleic, low linolenic, or lowsaturate oils (high oleic canola oil, low linolenic soybean oil or highstearic sunflower oils), animal fats (tallow, lard, butter fat, andchicken fat), fish oils (candlefish oil, cod-liver oil, orange roughyoil, sardine oil, herring oil, and menhaden oil), or blends of any ofthe above, and foods or baking, frying or cooking products, compriseoils with a lower saturated fatty acid content, including oils low inpalmitic acid, myristic acid, lauric acid, stearic acid, caprylic acid(octanoic acid) etc., processed by using a composition or method asprovided herein.

In one aspect, provided herein are polypeptides, for example, enzymesand catalytic antibodies, having a phospholipase activity, e.g.,phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC), including thermostable and thermotolerant enzymaticactivities, and fatty acid specific or fatty acid selective activities,and low or high pH tolerant enzymatic activities, and polynucleotidesencoding these polypeptides, including vectors, host cells, transgenicplants and non-human animals, and methods for making and using thesepolynucleotides and polypeptides.

In another aspect, provided herein are isolated, synthetic orrecombinant nucleic acids (a) encoding a polypeptide having aphospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) enzyme activity,and

-   -   (i) having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%,        82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to        SEQ ID NO:5 and encoding a polypeptide having at least one, two,        three, four, five, six, seven, eight, nine, ten, eleven, twelve,        thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,        nineteen, twenty, twenty-one, twenty-two, twenty-three,        twenty-four, twenty-five, twenty-six, twenty-seven,        twenty-eight, twenty-nine or thirty or more, or all, of the        amino acid changes (mutations) consisting of those described in        Table 12, Table 13, Table 14 and/or Table 15, or equivalent        amino acid substitutions or mutations, or any combination        thereof,    -   and optionally the sequence identities are determined by        analysis with a sequence comparison algorithm or by a visual        inspection,    -   and optionally the sequence comparison algorithm is a BLAST        version 2.2.2 algorithm where a filtering setting is set to        blastall -p blastp -d “nr pataa” -F F, and all other options are        set to default;    -   (ii) encoding a polypeptide have an amino acid sequence as set        forth in SEQ ID NO:6 and having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve, thirteen,        fourteen, fifteen, sixteen, seventeen, eighteen, nineteen,        twenty, twenty-one, twenty-two, twenty-three, twenty-four,        twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine        or thirty or more, or all, of the amino acid changes or        substitutions (mutations) consisting of those described in Table        12, Table 13, Table 14 and/or Table 15, or equivalent amino acid        changes or substitutions (mutations), or any combination        thereof; or    -   (iii) hybridizes under stringent conditions to a nucleic acid        comprising SEQ ID NO:5 and encoding a polypeptide having at        least one, two, three, four, five, six, seven, eight, nine, ten,        eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,        eighteen, nineteen, twenty, twenty-one, twenty-two,        twenty-three, twenty-four, twenty-five, twenty-six,        twenty-seven, twenty-eight, twenty-nine or thirty or more, or        all, of the amino acid changes (mutations) consisting of those        described in Table 12, Table 13, Table 14 and/or Table 15, or        equivalent amino acid changes or substitutions (mutations), or        any combination thereof,    -   wherein the stringent conditions comprise a wash step comprising        a wash in 0.2×SSC at a temperature of about 65° C. for about 15        minutes;    -   (iv) a nucleic acid comprising or consisting of the sequence SEQ        ID NO:7, SEQ ID NO:9 or SEQ ID NO:10; or    -   (v) having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%,        82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to        SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10;

(b) the nucleic acid sequence of (a) encoding a polypeptide having thephospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) enzyme activitybut lacking a native signal sequence or proprotein amino acid sequence;

(c) the nucleic acid sequence of (a) or (b) encoding a polypeptidehaving the phospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) enzyme activitybut lacking a native promoter sequence;

(d) the nucleic acid of (c) further comprising a heterologous promotersequence or other transcriptional regulatory sequence;

(e) the nucleic acid sequence of any of (a) to (d) further comprisingnucleic acid encoding a heterologous amino acid sequence, or furthercomprising a heterologous nucleotide sequence;

(f) the nucleic acid of (e), wherein the nucleic acid encoding theheterologous amino acid sequence comprises, or consists of, a sequenceencoding a heterologous (leader) signal sequence, or a tag or anepitope, or the heterologous nucleotide sequence comprises aheterologous promoter sequence;

(g) the nucleic acid of (d), (e) or (f), wherein the heterologousnucleotide sequence encodes a heterologous (leader) signal sequencecomprising or consisting of an N-terminal and/or C-terminal extensionfor targeting to an endoplasmic reticulum (ER) or endomembrane, or to aplant endoplasmic reticulum (ER) or endomembrane system, or theheterologous sequence encodes a restriction site;

(h) the nucleic acid of (d), (e) or (f), wherein the heterologouspromoter sequence comprises or consists of a constitutive or induciblepromoter, or a cell type specific promoter, or a plant specificpromoter, or a bacteria specific promoter;

(i) the nucleic acid of any of (a) to (h), wherein the phospholipase,e.g. a phospholipase C, e.g. a phosphatidylinositol-specificphospholipase C (PI-PLC) activity is thermostable;

(j) the nucleic acid of any of (a) to (h), wherein the phospholipase,e.g. a phospholipase C, e.g. a phosphatidylinositol-specificphospholipase C (PI-PLC) activity is thermotolerant;

(k) a nucleic acid sequence completely complementary to the nucleotidesequence of any of (a) to (j).

In one aspect, the isolated, synthetic or recombinant nucleic acidencodes a polypeptide or peptide having a phospholipase, e.g. aphospholipase C, e.g. a phosphatidylinositol-specific phospholipase C(PI-PLC) activity, which is thermostable. The polypeptides and peptidesencoded by nucleic acids as provided herein, or any polypeptide orpeptide as provided herein, can retain enzymatic or binding activity(e.g., substrate binding) under conditions comprising a temperaturerange of between about −100° C. to about −80° C., about −80° C. to about−40° C., about −40° C. to about −20° C., about −20° C. to about 0° C.,about 0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. toabout 25° C., about 25° C. to about 37° C., about 37° C. to about 45°C., about 45° C. to about 55° C., about 55° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 85° C., about 85° C. toabout 90° C., about 90° C. to about 95° C., about 95° C. to about 100°C., about 100° C. to about 105° C., 5 about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more. Provided herein are the thermostable polypeptides thatretain a phospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) activity, at atemperature in the ranges described above, at about pH 3.0, about pH3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH11.0, about pH 11.5, about pH 12.0 or more.

In one aspect, polypeptides as provided herein can be thermotolerant andcan retain a phospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) activity afterexposure to a temperature in the range from about −100° C. to about −80°C., about −80° C. to about −40° C., about −40° C. to about −20° C.,about −20° C. to about 0° C., about 0° C. to about 5° C., about 5° C. toabout 15° C., about 15° C. to about 25° C., about 25° C. to about 37°C., about 37° C. to about 45° C., about 45° C. to about 55° C., about55° C. to about 70° C., about 70° C. to about 75° C., about 75° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 105° C., about105° C. to about 110° C., about 110° C. to about 120° C., or 95° C., 96°C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C.,105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C.,113° C., 114° C., 115° C. or more.

In some embodiments, the thermotolerant polypeptides retain aphospholipase, e.g. a phospholipase C, e.g. aphosphatidylinositol-specific phospholipase C (PI-PLC) activity, afterexposure to a temperature in the ranges described above, at about pH3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

In another aspect, provided herein are nucleic acid probes oramplification primers for isolating, making and/or identifying a nucleicacid encoding a polypeptide having a phospholiplase, e.g. phospholipaseC, e.g. phosphatidylinositol-specific phospholipase C (PI-PLC) activity.In one embodiment, a nucleic acid probe, e.g., a probe for identifying anucleic acid encoding a polypeptide having a phospholiplase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity, comprises a probe comprising or consisting of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000 or more, consecutive bases of asequence as provided herein, or fragments or subsequences thereof,wherein the probe identifies the nucleic acid by binding orhybridization. The probe can comprise an oligonucleotide comprising atleast about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, orabout 60 to 100 consecutive bases of a sequence comprising a sequence asprovided herein, or fragments or subsequences thereof. The probe cancomprise an oligonucleotide comprising at least about 10 to 50, about 20to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutivebases of a nucleic acid sequence as provided herein, or a subsequencethereof.

In one embodiment, an amplification primer sequence pair for amplifyinga nucleic acid encoding a polypeptide having a phospholiplase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity, comprises a primer pair comprising or consisting of aprimer pair capable of amplifying a nucleic acid comprising a sequenceas provided herein, or fragments or subsequences thereof. One or eachmember of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 consecutive bases ofthe sequence.

In one embodiment, methods of amplifying a nucleic acid encoding apolypeptide having a phospholiplase, e.g. phospholipase C, e.g.phosphatidylinositol-specific phospholipase C (PI-PLC) activity,comprise amplification of a template nucleic acid with an amplificationprimer sequence air capable of amplifying a nucleic acid sequence asprovided herein, or fragments or subsequences thereof.

In one embodiment, vectors, expression cassettes, expression vectors,plasmids, or cloning vehicles comprise a nucleic acid as provided hereinor subsequence thereof. In one aspect, the vector, expression cassette,expression vector, plasmid, or cloning vehicle can comprise or iscontained in a viral vector, a phage, a phagemid, a cosmid, a fosmid, abacteriophage, an artificial chromosome, an adenovirus vector, aretroviral vector or an adeno-associated viral vector; or, a bacterialartificial chromosome (BAC), a bacteriophage P1-derived vector (PAC), ayeast artificial chromosome (YAC), or a mammalian artificial chromosome(MAC).

In one embodiment, expression cassettes comprise a nucleic acid asprovided herein or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

In one embodiment, a host cell or a transformed cell comprises a nucleicacid as provided herein. In one aspect, the host cell or a transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell. The transformedcell may be any of the host cells familiar to those skilled in the art,including prokaryotic cells, eukaryotic cells, such as bacterial cells,fungal cells, yeast cells, mammalian cells, insect cells, or plantcells. Exemplary bacterial cells include any species within the generaEscherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas andStaphylococcus, including, e.g., Escherichia coli, Lactococcus lactis,Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonasfluorescens. Exemplary fungal cells include any species of Aspergillus.Exemplary yeast cells include any species of Pichia, Saccharomyces,Schizosaccharomyces, or Schwanniomyces, including Pichia pastoris,Saccharomyces cerevisiae, or Schizosaccharomyces pombe. Exemplary insectcells include any species of Spodoptera or Drosophila, includingDrosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO,COS or Bowes melanoma or any mouse or human cell line.

In another embodiment, transgenic non-human animals comprise a nucleicacid as provided herein or a vector, expression cassette, expressionvector, plasmid, or cloning vehicle as provided herein. The transgenicnon-human animal can be a mouse, a rat, a goat, a rabbit, a sheep, a pigor a cow.

In one embodiment, a transgenic plant or seed comprises a nucleic acidas provided herein or a vector, expression cassette, expression vector,plasmid, or cloning vehicle as provided herein. In one embodiment, plantis a corn plant, a sorghum plant, a potato plant, a tomato plant, awheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a riceplant, a barley plant, a grass, a cottonseed, a palm, a sesame plant, apeanut plant, a sunflower plant or a tobacco plant; the transgenic seed.In one embodiment, the seed is a corn seed, a wheat kernel, an oilseed,a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesameseed, a rice, a barley, a peanut, a cottonseed, a palm, a peanut, asesame seed, a sunflower seed or a tobacco plant seed.

In one aspect, provided herein are an antisense oligonucleotide orinhibitory RNA comprising or consisting of a nucleic acid as providedherein.

In another aspect, provided herein is a method of inhibiting thetranslation of a phospholipase message (transcript, mRNA) in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide or inhibitory RNA comprising or consisting ofa nucleic acid sequence provided herein.

In one embodiment, isolated, synthetic or recombinant polypeptides havea phospholiplase, e.g. phospholipase C, e.g.phosphatidylinositol-specific phospholipase C (PI-PLC) activity, orpolypeptides capable of generating an immune response specific for aphospholiplase, e.g. phospholipase C, e.g. phosphatidylinositol-specificphospholipase C (PI-PLC) (e.g., an epitope); and in alternative aspectspeptides and polypeptides as provided herein comprise a sequence:

(a) comprising an amino acid sequence:

-   -   (i) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or more, or 100% sequence identity to SEQ ID        NO:6, and having at least one, two, three, four, five, six,        seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,        fifteen, sixteen, seventeen, eighteen, nineteen, twenty,        twenty-one, twenty-two, twenty-three, twenty-four, twenty-five,        twenty-six, twenty-seven, twenty-eight, twenty-nine or thirty or        more, or all, of the amino acid changes or substitutions        (mutations) consisting of those described in Table 12, Table 13,        Table 14 and/or Table 15, or equivalent amino acid changes or        substitutions (mutations), or any combination thereof,    -   wherein optionally the sequence identities are determined by        analysis with a sequence comparison algorithm or by a visual        inspection;    -   (ii) encoded by a nucleic acid as provided herein;    -   (iii) having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%,        82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or more, or 100% sequence identity to        SEQ ID NO:8;

(b) the polypeptide of (a) but lacking a native signal sequence and/or aproprotein sequence;

(c) the polypeptide of (a) or (b) further comprising a heterologousamino acid sequence or a heterologous moiety;

(d) the polypeptide of (c), wherein the heterologous amino acid sequenceor heterologous moiety comprises, or consists of a heterologous (leader)signal sequence, a tag, a detectable label or an epitope;

(e) the polypeptide of (d), wherein the heterologous (leader) signalsequence comprises or consisting of an N-terminal and/or C-terminalextension for targeting to an endoplasmic reticulum (ER) orendomembrane, or to a plant endoplasmic reticulum (ER) or endomembranesystem;

(f) the polypeptide of any of (a) to (e), wherein the phospholipase,e.g. phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) catalyzes a reaction comprising:1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate (also called PIP₂,phosphatidylinositol bisphosphate)+H₂O

1D-myo-inositol 1,4,5-trisphosphate (also called IP₃, inositoltriphosphate)+diacylglycerol;

(g) the polypeptide of (a) to (f), wherein the phospholipase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity is thermostable;

(h) the polypeptide of (a) to (f), wherein the phospholipase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity is thermotolerant;

(i) the polypeptide of any one of (a) to (h), wherein: (i) thepolypeptide is glycosylated, or the polypeptide comprises at least oneglycosylation site, (ii) the polypeptide of (i) wherein theglycosylation is an N-linked glycosylation or an O-linked glycosylation;(iii) the polypeptide of (i) or (ii) wherein the polypeptide isglycosylated after being expressed in a yeast cell; or (iv) thepolypeptide of (iii) wherein the yeast cell is a P. pastoris or a S.pombe;

(j) the polypeptide of any one of (a) to (i), further comprising orcontained in a composition comprising at least one second enzyme, or atleast one second phospholipase enzyme; or

(k) the polypeptide of (j), wherein the at least one secondphospholipase enzyme comprises a polypeptide having a sequence as setforth in SEQ ID NO:2 and/or SEQ ID NO:4, or at least one of theirvariant enzymes as described in Tables 8 and 9.

In one aspect, the isolated, synthetic or recombinant polypeptide cancomprise the polypeptide as provided herein that lacks a signal(peptide) sequence, e.g., lacks its homologous signal sequence, and inone aspect, comprises a heterologous signal (peptide) sequence. In oneaspect, the isolated, synthetic or recombinant polypeptide can comprisethe polypeptide as provided herein comprising a heterologous signalsequence, such as a heterologous phospholipase or non-phospholipase(e.g., non-phospholipase, non-phospholipase C ornon-phosphatidylinositol-specific phospholipase C (PI-PLC)) signalsequence. In one aspect, chimeric proteins comprise a first domaincomprising a signal sequence as provided herein and at least a seconddomain. The protein can be a fusion protein. The second domain cancomprise an enzyme. The enzyme can be a phospholipase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) as provided herein, or, another enzyme.

In one aspect, the phospholipase, e.g. phospholipase C, e.g.phosphatidylinositol-specific phospholipase C (PI-PLC) activitycomprises a specific activity at about 37° C. in the range from about100 to about 1000 units per milligram of protein. In another aspect, thephospholipase, e.g. phospholipase C, e.g. phosphatidylinositol-specificphospholipase C (PI-PLC) activity comprises a specific activity fromabout 500 to about 750 units per milligram of protein. Alternatively,the phospholipase activity comprises a specific activity at 37° C. inthe range from about 500 to about 1200 units per milligram of protein.In one aspect, the phospholipase activity comprises a specific activityat 37° C. in the range from about 750 to about 1000 units per milligramof protein. In another aspect, the thermotolerance comprises retentionof at least half of the specific activity of the phospholipase at 37° C.after being heated to an elevated temperature. Alternatively, thethermotolerance can comprise retention of specific activity at 37° C. inthe range from about 500 to about 1200 units per milligram of proteinafter being heated to an elevated temperature.

In one embodiment, the isolated, synthetic or recombinant polypeptidesas provided herein comprise at least one glycosylation site. In oneaspect, glycosylation can be an N-linked glycosylation. In one aspect,the polypeptide can be glycosylated after being expressed in a P.pastoris or a S. pombe or in plants, such as oil producing plants e.g.soy bean, canola, rice, sunflower, or genetically-modified (GMO)variants of these plants.

In one aspect, the polypeptide can retain a phospholipase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity under conditions comprising about pH 6.5, pH 6, pH5.5, pH 5, pH 4.5 or pH 4.0 or lower. In another aspect, the polypeptidecan retain a phospholipase, e.g. phospholipase C, e.g.phosphatidylinositol-specific phospholipase C (PI-PLC) activity underconditions comprising about pH 7, pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5, pH 11, pH 11.5, pH 12.0 or more.

In one embodiment, protein preparations comprise a polypeptide asprovided herein, wherein the protein preparation comprises a liquid, asolid or a gel.

In one aspect, heterodimers as provided herein comprise a polypeptideand a second domain. In one aspect, the second domain can be apolypeptide and the heterodimer can be a fusion protein. In one aspect,the second domain can be an epitope or a tag. In one aspect, homodimersas provided herein comprise a polypeptide as provided herein.

In one embodiment, immobilized polypeptides as provided herein have aphospholipase, e.g. phospholipase C, e.g. phosphatidylinositol-specificphospholipase C (PI-PLC) activity, wherein the polypeptide comprises apolypeptide as provided herein, a polypeptide encoded by a nucleic acidas provided herein, or a polypeptide comprising a polypeptide asprovided herein and a second domain. In one aspect, a polypeptide asprovided herein can be immobilized on a cell, a vesicle, a liposome, afilm, a membrane, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, a crystal,a tablet, a pill, a capsule, a powder, an agglomerate, a surface, aporous structure, an array or a capillary tube, or materials such asgrains, husks, bark, skin, hair, enamel, bone, shell and materialsderiving from them. Polynucleotides, polypeptides and enzymes asprovided herein can be formulated in a solid form such as a powder, alyophilized preparation, granules, a tablet, a bar, a crystal, acapsule, a pill, a pellet, or in a liquid form such as an aqueoussolution, an aerosol, a gel, a paste, a slurry, an aqueous/oil emulsion,a cream, a capsule, or a vesicular or micellar suspension.

In one aspect, provided herein are isolated, synthetic or recombinantantibodies which specifically binds to a polypeptide as provided herein.In another aspect, the isolated, synthetic or recombinant antibodies aremonoclonal or polyclonal antibodies, or are antigen binding fragmentsthereof. In one aspect, provided herein is an hybridoma comprising anantibody provided herein.

In one embodiment, provided herein is an array comprising an immobilizedpolypeptide, immobilized nucleic acid, or an antibody as providedherein, or a combination thereof.

In one embodiment, food supplements for an animal comprise a polypeptideas provided herein, e.g., a polypeptide encoded by the nucleic acid asprovided herein. In one aspect, the polypeptide in the food supplementcan be glycosylated. In one embodiment, edible enzyme delivery matricescomprise a polypeptide as provided herein, e.g., a polypeptide encodedby the nucleic acid as provided herein. In one aspect, the deliverymatrix comprises a pellet. In one aspect, the polypeptide can beglycosylated. In one aspect, the phospholipase activity isthermotolerant. In another aspect, the phospholipase activity isthermostable.

In one embodiment, methods of isolating or identifying a polypeptidehave a phospholipase, e.g. phospholipase C, e.g.phosphatidylinositol-specific phospholipase C (PI-PLC) activitycomprising the steps of: (a) providing an antibody as provided herein;(b) providing a sample comprising polypeptides; and (c) contacting thesample of step (b) with the antibody of step (a) under conditionswherein the antibody can specifically bind to the polypeptide, therebyisolating or identifying a polypeptide having a phospholipase, e.g.phospholipase C, e.g. phosphatidylinositol-specific phospholipase C(PI-PLC) activity.

In one embodiment, methods of making an anti-phospholipase antibodycomprise administering to a non-human animal a nucleic acid as providedherein or a polypeptide as provided herein or subsequences thereof in anamount sufficient to generate a humoral immune response, thereby makingan anti-phospholipase antibody. Provided herein are methods of making ananti-phospholipase antibody comprising administering to a non-humananimal a nucleic acid as provided herein or a polypeptide as providedherein or subsequences thereof in an amount sufficient to generate animmune response.

In one embodiment, methods of producing a recombinant polypeptidecomprise the steps of: (A) (a) providing a nucleic acid as providedherein, wherein the nucleic acid is optionally linked to a promoter,wherein the nucleic acid comprises a nucleic acid as provided herein;and (b) expressing the nucleic acid of step (a) under conditions thatallow expression of the polypeptide, thereby producing a recombinantpolypeptide; or (B) the method of (A), further comprising transforming ahost cell with the nucleic acid of step (a) followed by expressing thenucleic acid of step (a), thereby producing a recombinant polypeptide ina transformed cell.

In one embodiment, methods for identifying a polypeptide having aphospholipase activity comprise the steps of: (a) providing apolypeptide as provided herein; (b) providing a phospholipase substrate;and (c) contacting the polypeptide with the substrate of step (b) anddetecting a decrease in the amount of substrate or an increase in theamount of a reaction product, wherein a decrease in the amount of thesubstrate or an increase in the amount of the reaction product detects apolypeptide having a phospholipase activity.

In another embodiment, methods for identifying a phospholipase substratecomprise the steps of: (a) providing a polypeptide as provided herein;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as a phospholipase substrate.

In another aspect, methods of determining whether a test compoundspecifically binds to a polypeptide comprise the steps of: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid asprovided herein; (b) providing a test compound; (c) contacting thepolypeptide with the test compound; and (d) determining whether the testcompound of step (b) specifically binds to the polypeptide.

In another aspect, methods of determining whether a test compoundspecifically binds to a polypeptide comprise the steps of: (a) providinga polypeptide as provided herein; (b) providing a test compound; (c)contacting the polypeptide with the test compound; and (d) determiningwhether the test compound of step (b) specifically binds to thepolypeptide.

In one embodiment, methods for identifying a modulator of aphospholipase activity comprise the steps of: (A) (a) providing apolypeptide as provided herein; (b) providing a test compound; (c)contacting the polypeptide of step (a) with the test compound of step(b) and measuring an activity of the phospholipase, wherein a change inthe phospholipase activity measured in the presence of the test compoundcompared to the activity in the absence of the test compound provides adetermination that the test compound modulates the phospholipaseactivity; (B) the method of (A), wherein the phospholipase activity ismeasured by providing a phospholipase substrate and detecting a decreasein the amount of the substrate or an increase in the amount of areaction product, or, an increase in the amount of the substrate or adecrease in the amount of a reaction product; (c) the method of (B),wherein a decrease in the amount of the substrate or an increase in theamount of the reaction product with the test compound as compared to theamount of substrate or reaction product without the test compoundidentifies the test compound as an activator of phospholipase activity;or, (d) the method of (B), wherein an increase in the amount of thesubstrate or a decrease in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an inhibitorof phospholipase activity.

In one aspect, methods for isolating or recovering a nucleic acidencoding a polypeptide with a phospholipase activity from a samplecomprise the steps of: (A) (a) providing a polynucleotide probecomprising a nucleic acid as provided herein; (b) isolating a nucleicacid from the sample or treating the sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treated sampleof step (b) with the polynucleotide probe of step (a); and (d) isolatinga nucleic acid that specifically hybridizes with the polynucleotideprobe of step (a), thereby isolating or recovering a nucleic acidencoding a polypeptide with a phospholipase activity from a sample; (B)the method of (A), wherein the sample is or comprises an environmentalsample; (C) the method of (B), wherein the environmental sample is orcomprises a water sample, a liquid sample, a soil sample, an air sampleor a biological sample; or (D) the method of (C), wherein the biologicalsample is derived from a bacterial cell, a protozoan cell, an insectcell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.

In one embodiment, methods for isolating or recovering a nucleic acidencoding a polypeptide having a phospholipase activity from a samplecomprising the steps of: (a) providing an amplification primer sequencepair for amplifying a nucleic acid encoding a polypeptide having aphospholipase activity, wherein the primer pair is capable of amplifyinga nucleic acid as provided herein; (b) isolating a nucleic acid from thesample or treating the sample such that nucleic acid in the sample isaccessible for hybridization to the amplification primer pair; and, (c)combining the nucleic acid of step (b) with the amplification primerpair of step (a) and amplifying nucleic acid from the sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having aphospholipase activity from a sample. In one embodiment, the sample isan environmental sample, e.g., a water sample, a liquid sample, a soilsample, an air sample or a biological sample, e.g. a bacterial cell, aprotozoan cell, an insect cell, a yeast cell, a plant cell, a fungalcell or a mammalian cell. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 or more consecutive bases of a sequence as provided herein.

In one embodiment, methods of increasing thermotolerance orthermostability of a phospholipase polypeptide comprise glycosylating aphospholipase polypeptide, wherein the polypeptide comprises at leastthirty contiguous amino acids of a polypeptide as provided herein; or apolypeptide encoded by a nucleic acid sequence as provided herein,thereby increasing the thermotolerance or thermostability of thephospholipase polypeptide. In one aspect, the phospholipase specificactivity can be thermostable or thermotolerant at a temperature in therange from greater than about 37° C. to about 95° C.

In one embodiment, methods for overexpressing a recombinantphospholipase, e.g. phospholipase C, e.g. phosphatidylinositol-specificphospholipase C (PI-PLC) polypeptide in a cell comprise expressing avector comprising a nucleic acid as provided herein or a nucleic acidsequence as provided herein, wherein the sequence identities aredetermined by analysis with a sequence comparison algorithm or by visualinspection, wherein overexpression is effected by use of a high activitypromoter, a dicistronic vector or by gene amplification of the vector.

In one embodiment, methods for generating a variant of a nucleic acidencoding a polypeptide with a phospholipase activity comprise the stepsof: (A) (a) providing a template nucleic acid comprising a nucleic acidas provided herein; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid; (B) the method of (A),further comprising expressing the variant nucleic acid to generate avariant phospholipase polypeptide; (C) the method of (A) or (B), whereinthe modifications, additions or deletions are introduced by a methodcomprising error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR)and a combination thereof; (D) the method of any of (A) to (C), whereinthe modifications, additions or deletions are introduced by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof; (E) the method of any of(A) to (D), wherein the method is iteratively repeated until a (variant)phospholipase having an altered or different (variant) activity, or analtered or different (variant) stability from that of a polypeptideencoded by the template nucleic acid is produced, or an altered ordifferent (variant) secondary structure from that of a polypeptideencoded by the template nucleic acid is produced, or an altered ordifferent (variant) post-translational modification from that of apolypeptide encoded by the template nucleic acid is produced; (F) themethod of (E), wherein the variant phospholipase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature; (G) the method of (E), wherein the variantphospholipase polypeptide has increased glycosylation as compared to thephospholipase encoded by a template nucleic acid; (H) the method of (E),wherein the variant phospholipase polypeptide has a phospholipaseactivity under a high temperature, wherein the phospholipase encoded bythe template nucleic acid is not active under the high temperature; (I)the method of any of (A) to (H), wherein the method is iterativelyrepeated until a phospholipase coding sequence having an altered codonusage from that of the template nucleic acid is produced; or (J) themethod of any of (A) to (H), wherein the method is iteratively repeateduntil a phospholipase gene having higher or lower level of messageexpression or stability from that of the template nucleic acid isproduced.

In one aspect, methods for modifying codons in a nucleic acid encoding aphospholipase polypeptide, the method comprise the steps of: (a)providing a nucleic acid encoding a polypeptide with a phospholipaseactivity comprising a nucleic acid as provided herein; and, (b)identifying a codon in the nucleic acid of step (a) and replacing itwith a different codon encoding the same amino acid as the replacedcodon, thereby modifying codons in a nucleic acid encoding aphospholipase.

In one embodiment, methods for producing a library of nucleic acidsencoding a plurality of modified phospholipase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprise the steps of: (A) (a) providing a first nucleic acidencoding a first active site or first substrate binding site, whereinthe first nucleic acid sequence comprises a nucleic acid as providedherein, and the nucleic acid encodes a phospholipase active site or aphospholipase substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified phospholipase active sites or substrate bindingsites; (B) the method of (A), comprising mutagenizing the first nucleicacid of step (a) by a method comprising an optimized directed evolutionsystem, Gene Site Saturation Mutagenesis (GSSM), or a synthetic ligationreassembly (SLR); (C) the method of (A) or (B), comprising mutagenizingthe first nucleic acid of step (a) or variants by a method comprisingerror-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, Gene SiteSaturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) and acombination thereof; or (D) the method of (A) or (B), comprisingmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, methods for making a small molecule comprise the stepsof: (a) providing a plurality of biosynthetic enzymes capable ofsynthesizing or modifying a small molecule, wherein one of the enzymescomprises a phospholipase enzyme encoded by a nucleic acid as providedherein; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions.

In another aspect, methods for modifying a small molecule comprise thesteps of: (A) (a) providing a phospholipase enzyme, wherein the enzymecomprises a polypeptide as provided herein, or a polypeptide encoded bya nucleic acid as provided herein; (b) providing a small molecule; and(c) reacting the enzyme of step (a) with the small molecule of step (b)under conditions that facilitate an enzymatic reaction catalyzed by thephospholipase enzyme, thereby modifying a small molecule by aphospholipase enzymatic reaction; (B) the method of (A), comprising aplurality of small molecule substrates for the enzyme of step (a),thereby generating a library of modified small molecules produced by atleast one enzymatic reaction catalyzed by the phospholipase enzyme; (C)the method of (A) or (B), further comprising a plurality of additionalenzymes under conditions that facilitate a plurality of biocatalyticreactions by the enzymes to form a library of modified small moleculesproduced by the plurality of enzymatic reactions; (D) the method of (C),further comprising the step of testing the library to determine if aparticular modified small molecule which exhibits a desired activity ispresent within the library; or (E) the method of (D), wherein the stepof testing the library further comprises the steps of systematicallyeliminating all but one of the biocatalytic reactions used to produce aportion of the plurality of the modified small molecules within thelibrary by testing the portion of the modified small molecule for thepresence or absence of the particular modified small molecule with adesired activity, and identifying at least one specific biocatalyticreaction that produces the particular modified small molecule of desiredactivity.

In another aspect, methods for determining a functional fragment of aphospholipase enzyme comprise the steps of: (A) (a) providing aphospholipase enzyme, wherein the enzyme comprises a polypeptide asprovided herein, or a polypeptide encoded by a nucleic acid as providedherein; and (b) deleting a plurality of amino acid residues from thesequence of step (a) and testing the remaining subsequence for aphospholipase activity, thereby determining a functional fragment of aphospholipase enzyme; or, (B) the method of (A), wherein thephospholipase activity is measured by providing a phospholipasesubstrate and detecting a decrease in the amount of the substrate or anincrease in the amount of a reaction product.

In one aspect, methods for whole cell engineering of new or modifiedphenotypes by using real-time metabolic flux analysis, the methodcomprise the steps of: (a) making a modified cell by modifying thegenetic composition of a cell, wherein the genetic composition ismodified by addition to the cell of a nucleic acid as provided herein;(b) culturing the modified cell to generate a plurality of modifiedcells; (c) measuring at least one metabolic parameter of the cell bymonitoring the cell culture of step (b) in real time; and, (d) analyzingthe data of step (c) to determine if the measured parameter differs froma comparable measurement in an unmodified cell under similar conditions,thereby identifying an engineered phenotype in the cell using real-timemetabolic flux analysis; (B) the method of (A), wherein the geneticcomposition of the cell is modified by a method comprising deletion of asequence or modification of a sequence in the cell, or, knocking out theexpression of a gene; (C) the method of (A) or (B), further comprisingselecting a cell comprising a newly engineered phenotype; or (D) themethod of (C), further comprising culturing the selected cell, therebygenerating a new cell strain comprising a newly engineered phenotype.

In one embodiment, methods of making a transgenic plant comprise thefollowing steps: (a) introducing a heterologous nucleic acid sequenceinto a plant cell, wherein the heterologous nucleic sequence comprises anucleic acid sequence as provided herein, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

In one embodiment, methods of expressing a heterologous nucleic acidsequence in a plant cell comprise the following steps: (a) transformingthe plant cell with a heterologous nucleic acid sequence operably linkedto a promoter, wherein the heterologous nucleic sequence comprises anucleic acid as provided herein; (b) growing the plant under conditionswherein the heterologous nucleic acid sequence is expressed in the plantcell.

In one aspect, provided herein are detergent compositions comprising thephospholipase polypeptide as provided herein, or a phospholipasepolypeptide encoded by a nucleic acid as provided herein. In one aspect,the phospholipase is a nonsurface-active phospholipase or asurface-active phospholipase. In another aspect, the phospholipase isformulated in a non-aqueous liquid composition, a cast solid, alyophilized powder, a granular form, a particulate form, a compressedtablet, a pellet, a gel form, a paste, an aerosol, or a slurry form.

In one aspect, methods for washing an object comprise the steps of: (a)providing a composition comprising a phospholipase polypeptide asprovided herein, or a polypeptide encoded by a nucleic acid as providedherein; (b) providing an object; and (c) contacting the polypeptide ofstep (a) and the object of step (b) under conditions wherein thecomposition can wash the object.

In one embodiment, provided herein are compositions comprising aphospholipase polypeptide as provided herein, or a polypeptide encodedby a nucleic acid as provided herein.

In one aspect, methods for ameliorating, treating or preventinglipopolysaccharide (LPS)-mediated toxicity comprise administering to apatient a pharmaceutical composition comprising a polypeptide asprovided herein, or a polypeptide encoded by a nucleic acid sequence asprovided herein.

In another aspect, provided herein are pharmaceuticals, pharmaceuticalprecursors and pharmaceutical compositions comprising a polypeptide asprovided herein or a polypeptide encoded by a nucleic acid as providedherein. In another aspect, provided herein are methods of manufacturinga pharmaceutical, a pharmaceutical precursor or a pharmaceuticalcomposition comprising addition of a polypeptide encoded by a nucleicacid as provided herein to a pharmaceutical, a pharmaceutical precursoror a pharmaceutical composition. In one aspect, the pharmaceuticalcomposition is used for preventing, treating or amelioratinglipopolysaccharide (LPS)-mediated toxicity, or to detoxify an endotoxin,or deacylating a 2′ or a 3′ fatty acid chain from a lipid A.

In one embodiment, methods for detoxifying an endotoxin comprisecontacting the endotoxin with a polypeptide as provided herein or apolypeptide encoded by a nucleic acid as provided herein.

In another embodiment, methods for making a variant phospholipase codingsequence having increased expression in a host cell comprise modifying anucleic acid as provided herein, such that one, several or all N-linkedglycosylation site coding motifs are modified to a non-glycosylatedmotif.

In one embodiment, provided herein are compositions comprising a mixtureof phospholipase enzymes comprising: (a)(i) a phospholipase polypeptideas provided herein or polypeptide encoded by a nucleic acid as providedherein, and (ii) at least one second enzyme; (b) the composition of (a),wherein the at least one enzyme is a phospholipase enzyme; or (c) thecomposition of (b), wherein the at least one second phospholipase enzymecomprises a polypeptide as set forth in SEQ ID NO:2 and/or SEQ ID NO:4,or at least one of the variant PLC enzymes as described in Tables 8 and9.

In one aspect, methods for making a biofuel, e.g. a biodiesel, comprisethe steps of: (A) (a) providing a phospholipase polypeptide as providedherein, or a phospholipase enzyme encoded by a nucleic acid as providedherein, or a composition comprising a polypeptide as provided herein;(b) providing a composition comprising a lipid or an alkyl ester; (c)contacting the phospholipase polypeptide of (a) with the composition of(b); (B) the method of (A), wherein the composition comprising a lipidor an alkyl ester is, or comprises, an oil and/or a fat; or (C) themethod of (A) or (B), wherein the composition comprising a lipid or analkyl ester is, or comprises, an algae, a vegetable oil, a straightvegetable oil, a virgin vegetable oil, a waste vegetable oil, an animalfat, a grease, a tallow, a lard or a yellow grease. In another aspect,provided herein are fuels, e.g. biofuels, e.g. biodiesel, made bymethods that comprise the steps of: (A) (a) providing a phospholipasepolypeptide as provided herein, or a phospholipase enzyme encoded by anucleic acid as provided herein, or a composition comprising apolypeptide as provided herein; b) providing a composition comprising alipid or an alkyl ester; (c) contacting the phospholipase polypeptide of(a) with the composition of (b); (B) the method of (A), wherein thecomposition comprising a lipid or an alkyl ester is, or comprises, anoil and/or a fat; or (C) the method of (A) or (B), wherein thecomposition comprising a lipid or an alkyl ester is, or comprises, analgae, a vegetable oil, a straight vegetable oil, a virgin vegetableoil, a waste vegetable oil, an animal fat, a grease, a tallow, a lard ora yellow grease.

In another aspect, a distillers dried soluble (DDS), a distillers driedgrain (DDS), a condensed distillers soluble (CDS), a distillers wetgrain (DWG) or a distillers dried grain with solubles (DDGS), comprisesa polypeptide as provided herein, or a polypeptide encoded by a nucleicacid as provided herein, or a composition as provided herein.

In another embodiment, provided herein is a biomass comprising (a) apolypeptide as provided herein, or a polypeptide encoded by a nucleicacid as provided herein, or a composition as provided herein; (b) thebiomass of (a), wherein the biomass is, or comprises, an animal, algaeand/or plant biomass, or a lipid-comprising or lignocellulosic biomass,or a waste material.

In another embodiment, provided herein is a petroleum-based productcomprising: (a) a polypeptide as provided herein, or a polypeptideencoded by a nucleic acid as provided herein, or a composition asprovided herein; (b) made by a method comprising use of a polypeptide asprovided herein, or a polypeptide encoded by a nucleic acid as providedherein, or a composition as provided herein; or (c) the petroleum-basedproduct of (a) or (b) comprising an oil, a biodiesel or a gasoline, or abioethanol, biobutanol, biopropanol or a biomethanol; or a mixture ofbioethanol, biobutanol, biopropanol, biomethanol and/or biodiesel andgasoline.

In one embodiment, provided herein is a method for hydration of NonHydratable Phospholipids (NHPs) within a lipid matrix by enabling themto migrate to an oil-water interface. The NHPs are then reacted and/orremoved from the lipids. In one embodiment, the method comprises a)mixing an aqueous acid with an edible oil to obtain an acidic mixturehaving pH of less than about 4; and b) mixing a base with the acidicmixture to obtain a reacted mixture having pH of about 6-9. In certainembodiments, mixing in steps a) and/or b) creates an emulsion thatcomprises the aqueous phase in average droplet size between about 15 μmto about 45 μm. In certain embodiments, mixing in steps a) and/or b)creates an emulsion that comprises at least about 60% of the aqueousphase by volume in droplet size between about 15 μm to about 45 μm insize, wherein percentage of the aqueous phase is based on the totalvolume of the aqueous phase. In certain embodiments, mixing in steps a)and/or b) creates an emulsion that comprises at least about 60, 70, 80,90, 93, 95, 96, 97, 98, or 99% of the aqueous phase by volume in dropletsize between about 20 μm to about 45 μm in size. In certain embodiments,the method further comprises degumming the reacted mixture with water oran enzyme to obtain a degummed oil. In certain embodiments, the mixingin steps a) and/or b) is carried out with a high shear mixer with a tipspeed of at least about 1400 cm/s, 1600 cm/s, 1800 cm/s, 2000 cm/s, 2100cm/s, 2300 cm/s, 2500 cm/s, 3000 cm/s, or 3500 cm/s.

Any acid deemed suitable by one of skill in the art can be used in themethods provided herein. In certain embodiments, the acid is selectedfrom the group consisting of phosphoric acid, acetic acid, citric acid,tartaric acid, succinic acid, and a mixture thereof. Any acid deemedsuitable by one of skill in the art can be used in the methods providedherein. In certain embodiments, the base is selected from the groupconsisting of sodium hydroxide, potassium hydroxide, sodium silicate,sodium carbonate, calcium carbonate, and a combination thereof.

In certain embodiments, the method for hydration of non hydratablephospholipids in an edible oil further comprises a step of water orenzymatic degumming to obtain a degummed oil. In one embodiment,provided herein is a method wherein NHPs hydration is followed byenzymatic treatment and removal of various phospholipids and lecithins.Such methods can be practiced on either crude or water-degummed oils.

In certain embodiments, an oil degumming method provided hereincomprises a) mixing an aqueous acid with an edible oil to obtain anacidic mixture having pH of about 1 to 4, b) mixing a base with theacidic mixture to obtain a reacted mixture having pH of about 6-9, andc) degumming the reacted mixture with water or an enzyme to obtain adegummed oil. In certain embodiments, mixing in steps a) and/or b)creates an emulsion that comprises an aqueous phase in average dropletsize between about 15 μm to about 45 μm. In certain embodiments, mixingin steps a) and/or b) creates an emulsion that comprises at least about60% of an aqueous phase by volume in droplet size between about 15 μm toabout 45 μm in size, wherein percentage of the aqueous phase is based onthe total volume of the aqueous phase.

In one embodiment, provided herein is a method for removing NHPs,hydratable phospholipids, and lecithins (known collectively as “gums”)from vegetable oils to produce a degummed oil or fat product that can beused for food production and/or non-food applications. In certainembodiments, the degumming methods provided herein utilize water,various acids and/or various bases or a combination thereof.

In another aspect, provided herein is a method for enhancing thereaction rate of a phospholipase used in an enzymatic degumming method,such that the enzyme reaction has a duration of less than about onehour.

In yet another aspect, provided herein is a method for degumming an oilcomposition in which both hydratable and non-hydratable phospholipidscan be treated in a single process, wherein an enzyme reaction iscompleted in less than about one hour.

In one embodiment, provided herein is a method for hydrolyzing, breakingup or disrupting a phospholipid-comprising composition comprising:

(A) (a) providing a phospholipase polypeptide; (b) providing acomposition comprising a phospholipid; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the phospholipase hydrolyzes, breaks up or disruptsthe phospholipid-comprising composition;

(B) the method of (A), wherein the composition comprises aphospholipid-comprising lipid bilayer or membrane; or

(C) the method of any of (A) or (B), wherein the composition comprises aplant cell, a bacterial cell, a yeast cell, an insect cell, or an animalcell.

In one embodiment, provided herein is a method for liquefying orremoving a phospholipid-comprising composition comprising:

(a) providing a phospholipase polypeptide;

(b) providing a composition comprising a phospholipid; and

(c) contacting the polypeptide of step (a) with the composition of step(b) under conditions wherein the phospholipase removes or liquefies thephospholipid-comprising composition.

In one embodiment, provided herein is a method for purification of aphytosterol or a triterpene comprising:

-   -   AI) (AIa) providing a composition comprising a phospholipase        polypeptide;    -   (AIb) providing an composition comprising a phytosterol or a        triterpene; and    -   (AIc) contacting the polypeptide of step (a) with the        composition of step (b) under conditions wherein the polypeptide        can catalyze the hydrolysis of a phospholipid in the        composition;    -   (BI) the method of (AI), wherein the phytosterol or a triterpene        comprises a plant sterol;    -   (CI) the method of (BI), wherein the plant sterol is derived        from a vegetable oil;    -   (DI) the method of (CI), wherein the vegetable oil comprises a        coconut oil, canola oil, cocoa butter oil, corn oil, cottonseed        oil, linseed oil, olive oil, palm oil, peanut oil, oil derived        from a rice bran, safflower oil, sesame oil, soybean oil or a        sunflower oil;    -   (EI) the method of any of (AI) to (DI), further comprising use        of nonpolar solvents to quantitatively extract free phytosterols        and phytosteryl fatty-acid esters; or    -   (FI) the method of (EI), wherein the phytosterol or a triterpene        comprises a β-sitosterol, a campesterol, a stigmasterol, a        stigmastanol, a β-sitostanol, a sitostanol, a desmosterol, a        chalinasterol, a poriferasterol, a clionasterol or a        brassicasterol.

In one embodiment, provided herein is a method for refining an oil or afat comprising:

-   -   (A1) (A1a) providing a composition comprising a phospholipase        polypeptide;    -   (A1b) providing a composition comprising an oil or a fat        comprising a phospholipid; and    -   (A1c) contacting the polypeptide of step (A1a) with the        composition of step (A1b) under conditions wherein the        polypeptide can catalyze the hydrolysis of a phospholipid in the        composition;    -   (B1) the method of (A1), wherein the polypeptide is in a water        solution that is added to the composition;    -   (C1) the method of (B1), wherein the water level is between        about 0.5 to 5%;    -   (D1) the method of any of (A1) to (C1), wherein the process time        is less than about 2 hours;    -   (E1) the method of any of (A1) to (C1), wherein the process time        is less than about 60 minutes;    -   (F1) the method of any of (A1) to (C1), wherein the process time        is less than about 30 minutes, less than about 15 minutes, or        less than about 5 minutes;    -   (G1) the method of any of (A1) to (F1), wherein the hydrolysis        conditions comprise a temperature of between about 25° C. to 70°        C.;    -   (H1) the method of any of (A1) to (G1), wherein the hydrolysis        conditions comprise use of caustics;    -   (I1) the method of any of (A1) to (H1), wherein the hydrolysis        conditions comprise a pH of between about pH 3 and pH 10;    -   (J1) the method of any of (A1) to (I1), wherein the hydrolysis        conditions comprise addition of emulsifiers and/or mixing after        the contacting of step (A1) (A1c);    -   (K1) the method of any of (A1) to (J1), comprising addition of        an emulsion-breaker and/or heat or cooling to promote separation        of an aqueous phase;    -   (L1) the method of any of (A1) to (K1), comprising degumming        before the contacting step to collect lecithin by centrifugation        and then adding a PLC, a PLC and/or a PLA to remove        non-hydratable phospholipids;    -   (M1) the method of any of (A1) to (L1), comprising water        degumming of crude oil to less than 10 ppm phosphorus for edible        oils and subsequent physical refining to less than about 50 ppm        phosphorus for biodiesel oils; or    -   (N1) the method of any of (A1) to (M1), comprising addition of        acid to promote hydration of non-hydratable phospholipids.

In one embodiment, provided herein is a method for degumming an oil or afat comprising

-   -   (a1) providing a composition comprising a phospholipase        polypeptide;    -   (b1) providing an composition comprising an        phospholipid-containing fat or oil; and    -   (c1) contacting the polypeptide of step (a1) and the composition        of step (b1) under conditions wherein the polypeptide can        catalyze the hydrolysis of a phospholipid in the composition.

In one embodiment, provided herein is a method for physical refining ofa phospholipid-containing composition comprising:

-   -   (A-1) (A-1a) providing a composition comprising a phospholipase        polypeptide;    -   (A-1b) providing an composition comprising a phospholipid; and    -   (A-1c) contacting the polypeptide of step (A-1a) with the        composition of step (A-1b) before, during or after the physical        refining;    -   (B-1) the method of (A-1), wherein the polypeptide is added        before physical refining and the composition comprising the        phospholipid comprises a plant and the polypeptide is expressed        transgenically in the plant, the polypeptide is added during        crushing of a seed or other plant part, or, the polypeptide is        added following crushing or prior to refining;    -   (C-1) the method of (A-1), wherein the polypeptide is added        during the physical refining;    -   (D-1) the method of (A-1), wherein the polypeptide is added        after physical refining: in an intense mixer or retention mixer        prior to separation; following a heating step; in a centrifuge;        in a soapstock; in a washwater; or, during a bleaching or a        deodorizing step; or    -   (E-1) the method of any of (A-1) to (D-1), further comprising        adding a phospholipase A (PLA), a phospholipase B (PLB),        phospholipase C (PLC), phospholipase D (PLD), or a phosphatase        enzyme, or any combination thereof.

In one embodiment, provided herein is a method for caustic refining of aphospholipid-containing composition comprising:

(A1) (A1a) providing a composition comprising a polypeptide having aphospholipase activity;

-   -   (A1b) providing an composition comprising a phospholipid; and    -   (A1c) contacting the polypeptide of step (A1a) with the        composition of step (A1b) before, during or after the caustic        refining;

(B1) the method of (A1), wherein the polypeptide is added beforeaddition of acid or caustic;

(C1) the method of any of (A1) to (B1), wherein the polypeptide is addedduring caustic refining and varying levels of acid and caustic are addeddepending on levels of phosphorus and levels of free fatty acids; or

(D1) the method of any of (A1) to (B1), wherein the polypeptide is addedafter caustic refining: in an intense mixer or retention mixer prior toseparation; following a heating step; in a centrifuge; in a soapstock;in a washwater; or, during bleaching or deodorizing steps;

(E1) the method of any of (A1) to (D1), wherein caustic refiningconditions are generated by addition of a concentrated solution ofcaustic, or wherein caustic refining conditions comprise use of aconcentrated solution of caustic more concentrated than the industrialstandard of 11%, or wherein caustic refining conditions comprise use ofa concentrated solution of caustic that is between about 12% and 50%concentrated;

(F1) the method of any of (A1) to (E1), wherein the compositioncomprising the phospholipid comprises a plant;

(G1) the method of any of (F1), wherein the polypeptide is expressedtransgenically in the plant;

(H1) the method of any of (A1) to (G1), wherein the polypeptide is addedduring crushing of a seed or other plant part, or, the polypeptide isadded following crushing or prior to refining; or

(I1) the method of any of (A1) to (H1), comprising a process as setforth in FIG. 10; or the process as set forth in FIG. 10, whereinsufficient acid is added to promote lowering of the calcium andmagnesium metal content.

In one embodiment, provided herein is a method for deacylating a 2′ or a3′ fatty acid chain from a lipid A comprising contacting the lipid Awith a phospholipase polypeptide.

In one embodiment, provided herein is a process for reducing gum massand increasing neutral oil (triglyceride) gain through reduced oilentrapment comprising:

-   -   (A1) (A1a) providing a composition comprising a phospholipase        polypeptide;        -   (A1b) providing an composition comprising an            phospholipid-containing fat or oil; and        -   (A1c) contacting the polypeptide of step (A1a) and the            composition of step (A1b) under conditions wherein the            polypeptide can catalyze the hydrolysis of a phospholipid in            the composition for a time sufficient to reduce gum mass and            increase neutral oils;    -   (B1) the protein preparation of (A1), wherein the protein        preparation comprises a formulation comprising a non-aqueous        liquid composition, a cast solid, a powder, a lyophilized        powder, a granular form, a particulate form, a compressed        tablet, a pellet, a pill, a gel form, a hydrogel, a paste, an        aerosol, a spray, a lotion, a slurry formulation, an aqueous/oil        emulsion, a cream, a capsule, a vesicle, or a micellar        suspension; or,    -   (C1) the method of (A1) or (B1), comprising use of high shear        mixing of the composition, followed by no or low shear mixing        with the at least one polypeptide of the invention having a        phospholipase activity to allow adequate contacting of the        phospholipid substrate with the phospholipase.

In one embodiment, provided herein is an oil or fat produced by themethods provided herein.

The enzymes for use in the methods provided herein include enzymeshaving phospholipase activity. The phospholipase activity comprises, forexample, a phospholipase C (PLC) activity, a phospholipase A (PLA)activity, including a phospholipase A1 or phospholipase A2 activity, aphospholipase B (PLB) activity, including a phospholipase B1 orphospholipase B2 activity, a phospholipase D (PLD) activity, including aphospholipase D1 or a phospholipase D2 activity. In one embodiment, theenzymes for use herein comprise polypeptides having aphosphatidylinositol-specific phospholipase C (PI-PLC) or equivalentenzyme activity, and/or another phospholipase activity, including aphospholipase A, B, C, D, patatin, phosphatidic acid phosphatases (PAP)and/or lipid acyl hydrolase (LAH) or equivalent enzyme activity.

In certain embodiments, the enzyme for use in the methods providedherein is selected from a phospholipase A, phospholipase C,phosphatidyl-inositol specific phospholipase C, or a combinationthereof. In certain embodiments, the enzyme for use in the methodsprovided herein is selected from a phospholipase C,phosphatidyl-inositol specific phospholipase C, or a combinationthereof. In certain embodiments, the enzyme for use in the methodsprovided herein is phosphatidyl-inositol specific phospholipase C enzymeas described elsewhere herein. In certain embodiments, the enzyme foruse in the methods provided herein is selected from phospholipase C, andan enzyme comprising SEQ ID NO:8. In certain embodiments, the enzyme foruse in the methods provided herein is an enzyme comprising SEQ ID NO:8.

In another embodiment, provided herein is a method for obtaining aphospholipid from an edible oil. In certain embodiment, thephospholipids obtained by the methods provided herein include a varietyof phospholipids, including, but not limited to phosphatidylcholine(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylinositol (PI), phosphatidic acid (PA),lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE),lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI),lysophosphatidic acid (LPA), choline (C), ethanolamine (E), serine (S),and inositol (I).

In another embodiment, provided herein is a method for obtaining choline(C), ethanolamine (E), serine (S), or inositol (I) from an edible oil.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 schematically illustrates an exemplary vegetable oil refiningprocess using the phospholipases of the invention.

FIG. 2 schematically illustrates an exemplary degumming process of theinvention for physically refined oils, as discussed in detail, below.

FIG. 3 schematically illustrates phosphatide hydrolysis with aphospholipase C of the invention, as discussed in detail, below.

FIG. 4 schematically illustrates an exemplary caustic refining processof the invention, and illustrates an alternative embodiment comprisingapplication of a phospholipase C of the invention as a “Caustic RefiningAid” (Long Mix Caustic Refining), as discussed in detail, below.

FIG. 5 schematically illustrates application of a phospholipase C of theinvention as a degumming aid, as discussed in detail, below.

FIG. 6 schematically illustrates an exemplary caustic refining processof the invention, and illustrates an alternative embodiment comprisingapplication of a phospholipase C of the invention as a “Caustic RefiningAid” (Long Mix Caustic Refining), as discussed in detail, below.

FIG. 7 illustrates another variation of methods of the invention wheretwo centrifugation steps are used in the process, as discussed indetail, below.

FIG. 8 illustrates another variation of methods of the invention wherethree centrifugation steps are used in the process, as discussed indetail, below.

FIG. 9 illustrates another exemplary variation of this process usingacid treatment and having a centrifugation step before a degumming step,as discussed in detail, below.

FIG. 10 illustrates the weight-fraction of individual phospholipid (PL)species phosphatidic acid (PA), phosphatidylethanolamine (PE),phosphatidylinositol (PI), phosphatidylcholine (PC) relative to thetotal PL remaining after treatment with the mutant phospholipases of theinvention.

FIG. 11 illustrates the “Gene Site Saturation Mutagenesis” or “GSSM”upmutants selected for inclusion in the GeneReassembly Library, whichincludes exemplary phospholipases of the invention.

FIG. 12 illustrates an exemplary alcohol process that can incorporateuse of enzymes of this invention.

FIG. 13 provides phospholipid composition of the recovered wet gums inthe control examples.

FIG. 14 provides phospholipid comparisons for the control examplesversus the examples using the methods provided herein where pH in stepb) is adjusted to pH 7.0.

FIG. 15 compares the neutral oil lost to the gum phase in the controlneutral pH reactions versus the pH adjusted reactions at a pH of 7.0,side-by-side.

FIG. 16 compares the neutral oil lost to the gum phase of various pHconditions where a phospholipase A 1 is utilized.

FIG. 17 depicts droplet distribution for aqueous phase obtainedaccording to the process of Example 17A.

FIG. 18 depicts droplet distribution for aqueous phase obtainedaccording to the process of Example 17B.

FIG. 19 depicts droplet distribution for aqueous phase obtainedaccording to the process of Example 17C.

FIG. 20 depicts droplet distribution for aqueous phase obtainedaccording to the process of Example 17D.

FIG. 21 depicts comparative droplet distribution for aqueous phaseobtained according to the processes of Examples 17A, 17B, 17C and 17D.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides having aphosphatidylinositol-specific phospholipase C (PI-PLC) or equivalentenzyme activity, and/or another phospholipase activity, including aphospholipase A, B, C, D, patatin, phosphatidic acid phosphatases (PAP)and/or lipid acyl hydrolase (LAH) or equivalent enzyme activity,polynucleotides encoding them, antibodies that bind specifically tothem, and methods for making and using them.

In one embodiment, the phosphatidylinositol-specific phospholipase C(PI-PLC) enzyme activity of polypeptides of this invention comprise:1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate (also called PIP₂,phosphatidylinositol bisphosphate)+H₂O

1D-myo-inositol 1,4,5-trisphosphate (also called IP₃, inositoltriphosphate)+diacylglycerol.

In alternative embodiments, enzymes of the invention can efficientlycleave glycerolphosphate ester linkage in oils, such as vegetable oils,e.g., oilseed phospholipids, to generate a water extractablephosphorylated base and a diglyceride.

In alternative embodiments, phospholipases of the invention have a lipidacyl hydrolase (LAH) activity; or can cleave glycerolphosphate esterlinkages in phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid,and/or sphingomyelin, or a combination of these activities. For example,in one aspect a phospholipase of the invention is specific for one ormore specific substrates, e.g., an enzyme of the invention can have aspecificity of action for PE and PC; PE an PI; PE and PS; PS and PC; PSand PI; PI and PC; PS, PI and PC; PE, PI and PC; PC, PE and PS; PE, PSand PI; or, PE, PS, PI and PC.

In alternative embodiments, a phospholipase of the invention (e.g., aphosphatidylinositol-specific phospholipase C (PI-PLC) enzyme orequivalent activity) can be used for enzymatic degumming of oils, e.g.crude oils, because the phosphate moiety is soluble in water and easy toremove. The diglyceride product will remain in the oil and thereforewill reduce losses. The PLCs of the invention can be used in addition toor in place of PLA1s and PLA2s in commercial oil degumming, such as inthe ENZYMAX® process, where phospholipids are hydrolyzed by PLA1 andPLA2.

In alternative embodiments, the phospholipases of the invention areactive at a high and/or at a low temperature, or, over a wide range oftemperature, e.g., they can be active in the temperatures rangingbetween 20° C. to 90° C., between 30° C. to 80° C., or between 40° C. to70° C. The invention also provides phospholipases that can have activityat alkaline pHs or at acidic pHs, e.g., low water acidity. Inalternative aspects, the phospholipases of the invention can haveactivity in acidic pHs as low as pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5,pH 4.0, pH 3.5, pH 3.0, pH 2.5, pH 2.0 or more acidic (i.e., <pH 2.0).In alternative aspects, the phospholipases of the invention can haveactivity in alkaline pHs as high as pH 7.5, pH 8.0, pH 8.5, pH 9.0, pH9.5, pH 10.0 or more alkaline (i.e., >pH 10.0). In one aspect, thephospholipases of the invention are active in the temperature range ofbetween about 40° C. to about 70° C., 75° C., or 80° C., or more, underconditions of low water activity (low water content).

The invention also provides methods for making PLCs and/or modifying theactivity of exemplary phospholipases of the invention to generateenzymes with alternative desirable properties, e.g.,phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme activityhaving alternative substrates, or activities under various environmentalconditions, e.g., of varying temperatures, pHs and the like. Forexample, phospholipases generated by the methods of the invention canhave altered substrate specificities, substrate binding specificities,substrate cleavage patterns, thermal stability, pH/activity profile,pH/stability profile (such as increased stability at low, e.g. pH<6 orpH<5, or high, e.g. pH>9, pH values), stability towards oxidation, Ca²⁺dependency, specific activity and the like. The invention provides foraltering any property of interest. For instance, the alteration mayresult in a variant which, as compared to a parent phospholipase, hasaltered pH and temperature activity profile.

In alternative embodiments, the phospholipases of the invention are usedin various oil processing steps, such as in oil extraction, e.g., in theremoval of “phospholipid gums” in a process called “oil degumming,” asdescribed herein. The invention provides compositions (e.g., comprisingenzymes of the invention) and processes for the treatment of oils, e.g.crude oils, and for production of oils, e.g. vegetable oils, fromvarious sources, such as oil from rice bran, soybeans, rapeseed, peanut,sesame, sunflower and corn. The phospholipase enzymes of the inventioncan be used in place of PLA, e.g., phospholipase A2, in any vegetableoil processing step.

In certain embodiments, suitable enzymes for use in the methods providedherein include, one or more phospholipase A (PLA) enzymes, phospholipaseC (PLC), Phosphatidyl-Inositol specific phospholipase C (PI-PLC)enzymes, or a combination thereof. The PLA enzymes include phospholipaseA1 (PLA1) and/or phospholipase A2 (PLA2).

As used herein, “crude oil” refers to (also called a non-degummed oil) apressed or extracted oil or a mixture thereof from, e.g. vegetablesources, including but not limited to acai oil, almond oil, babassu oil,blackcurrent seed oil, borage seed oil, canola oil, cashew oil, castoroil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil,flax seed oil, grape seed oil, hazelnut oil, hempseed oil, jatropha oil,jojoba oil, linseed oil, macadamia nut oil, mango kernel oil, meadowfoamoil, mustard oil, neat's foot oil, olive oil, palm oil, palm kernel oil,palm olein, peanut oil, pecan oil, pine nut oil, pistachio oil, poppyseed oil, rapeseed oil, rice bran oil, safflower oil, sasanqua oil,sesame oil, shea butter, soybean oil, sunflower seed oil, tall oil,tsubaki oil walnut oil, varieties of “natural” oils having altered fattyacid compositions via Genetically Modified Organisms (GMO) ortraditional “breading” such as high oleic, low linolenic, or lowsaturated oils (high oleic canola oil, low linolenic soybean oil or highstearic sunflower oils). Further exemplary oils suitable for use in themethods provided herein are described elsewhere herein. In certainembodiment, the total phosphatide content in a crude oil may vary from0.5-3% w/w corresponding to a phosphorus content in the range of200-1200 ppm or 250-1200 ppm.

As used herein, “degummed oil” refers to an oil obtained after removalof NHPs, hydratable phospholipids, and lecithins (known collectively as“gums”) from the oil to produce a degummed oil or fat product that canbe used for food production and/or non-food applications. Variousdegumming process are known in the art and are described above. Incertain embodiments, the degummed oil has the phospholipids content ofless than about 200 ppm phosphorus, less than about 150 ppm phosphorus,less than about 100 ppm phosphorus, less than about 50 ppm phosphorus,less than about 40 ppm phosphorus, less than about 30 ppm phosphorus,less than about 20 ppm phosphorus, less than about 15 ppm phosphorus,less than about 10 ppm phosphorus, less than about 7 ppm phosphorus,less than about 5 ppm phosphorus, less than about 3 ppm phosphorus orless than about 1 ppm phosphorus.

As used herein, the term “non hydratable phospholipids” or “NHPs” refersto phosphatidic acid and the salts of phosphatidic acid, for example,calcium, magnesium and iron salts of phosphatidic acid; and calcium,magnesium and iron salts of ethanolamine.

As used herein, “water-degummed oil” refers to an oil obtained afterwater degumming process. In certain embodiments, water-degummed oil isobtained by mixing 1-3% w/w of hot water with warm (60-90° C.) crude oilfor 30-60 minutes. In certain embodiments, the water-degumming stepremoves the phosphatides and mucilaginous gums which become insoluble inthe oil when hydrated. The hydrated phosphatides and gums can beseparated from the oil by settling, filtering or centrifuging.

Generating and Manipulating Nucleic Acids

The invention provides isolated, synthetic and recombinant nucleic acids(e.g., the exemplary SEQ ID NO:5 and nucleic acids encoding SEQ ID NO:6comprising (and having) one or more amino acid residue changes (e.g.,mutations) as set forth in Tables 12 to 15, including expressioncassettes such as expression vectors, encoding the polypeptides andphospholipases of the invention. The invention also includes methods fordiscovering new phospholipase sequences using the nucleic acids of theinvention. Also provided are methods for modifying the nucleic acids ofthe invention by, e.g., synthetic ligation reassembly, optimizeddirected evolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

In alternative embodiments, gene sequences of the invention, or genesused to practice the invention, comprise the segment of DNA involved inproducing a polypeptide chain, including, inter alia, regions precedingand following the coding region, such as leader and trailer, promotersand enhancers, as well as, where applicable, intervening sequences(introns) between individual coding segments (exons).

In alternative embodiments, nucleic acids or nucleic acid sequences ofthe invention, or used to practice the invention, can comprise anoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., double stranded iRNAs,e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Inalternative embodiments, nucleic acids or nucleic acid sequences of theinvention, or used to practice the invention, also encompassnucleic-acid-like structures with synthetic backbones, see e.g., Mata(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997)Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid DrugDev 6:153-156.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Expression Cassettes, Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thephospholipases of the invention. Expression vectors and cloning vehiclesof the invention can comprise viral particles, baculovirus, phage,plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes,viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as Bacillus, Aspergillus and yeast).Vectors of the invention can include chromosomal, non-chromosomal andsynthetic DNA sequences. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Exemplaryvectors are include: bacterial: pQE vectors (Qiagen, San Diego, Calif.),pBluescript plasmids (Stratagene, San Diego, Calif.), pNH vectors,(lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG,pSVLSV40 (Pharmacia). However, any other plasmid or other vector may beused so long as they are replicable and viable in the host. Low copynumber or high copy number vectors may be employed with the presentinvention.

In alternative embodiments, the term “expression cassette” comprises anucleotide sequence which is capable of affecting expression of astructural gene (i.e., a protein coding sequence, such as aphospholipase of the invention) in a host compatible with suchsequences. Expression cassettes can include at least a promoter operablylinked with the polypeptide coding sequence; and, optionally, with othersequences, e.g., transcription termination signals. Additional factorsnecessary or helpful in effecting expression may also be used, e.g.,enhancers. In alternative embodiments, “operably linked” refers tolinkage of a promoter upstream from a DNA sequence such that thepromoter mediates transcription of the DNA sequence. In alternativeembodiments, expression cassettes include plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike.

In alternative embodiments, vectors of this invention comprise a nucleicacid which can infect, transfect, transiently or permanently transduce acell. In alternative embodiments, a vector can be a naked nucleic acid,or a nucleic acid complexed with protein or lipid. The vector optionallycomprises viral or bacterial nucleic acids and/or proteins, and/ormembranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectorsof this invention include, but are not limited to replicons (e.g., RNAreplicons, bacteriophages) to which fragments of DNA may be attached andbecome replicated. Vectors thus include, but are not limited to RNA,autonomous self-replicating circular or linear DNA or RNA (e.g.,plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879),and includes both the expression and non-expression plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector” this includes both extra-chromosomal circular andlinear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

In alternative embodiments, the invention provides plasmids, which canbe designated by a lower case “p” preceded and/or followed by capitalletters and/or numbers. In alternative embodiments, a “starting” plasmidis either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In alternative embodiments, equivalent plasmids tothose described herein are known in the art and will be apparent to theordinarily skilled artisan.

In alternative embodiments, an expression vector may comprise apromoter, a ribosome-binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying expression. Mammalian expression vectors cancomprise an origin of replication, any necessary ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. Insome aspects, DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures.In general, the DNA sequence is ligated to the desired position in thevector following digestion of the insert and the vector with appropriaterestriction endonucleases. Alternatively, blunt ends in both the insertand the vector may be ligated. A variety of cloning techniques are knownin the art, e.g., as described in Ausubel and Sambrook. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The vector may be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a phospholipase ofthe invention, a vector of the invention. The host cell may be any ofthe host cells familiar to those skilled in the art, includingprokaryotic cells, eukaryotic cells, such as bacterial cells, fungalcells, yeast cells, mammalian cells, insect cells, or plant cells.Enzymes of the invention can be expressed in any host cell, e.g., anybacterial cell, any yeast cell, e.g., Pichia pastoris, Saccharomycescerevisiae or Schizosaccharomyces pombe. Exemplary bacterial cellsinclude any species within the genera Escherichia, Bacillus,Streptomyces, Salmonella, Pseudomonas and Staphylococcus, including,e.g., Escherichia coli, Lactococcus lactis, Bacillus subtilis, Bacilluscereus, Salmonella typhimurium, Pseudomonas fluorescens. Exemplaryfungal cells include any species of Aspergillus. Exemplary yeast cellsinclude any species of Pichia, Saccharomyces, Schizosaccharomyces, orSchwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe. Exemplary insect cells include any species ofSpodoptera or Drosophila, including Drosophila S2 and Spodoptera Sf9.Exemplary animal cells include CHO, COS or Bowes melanoma or any mouseor human cell line. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

An exemplary PI-PLC enzyme (having a sequence as set forth in SEQ IDNO:6 comprising (and having) one or more amino acid residue changes(e.g., mutations) as set forth in Tables 12 to 15) has beenover-expressed in active form in a variety of host systems includinggram negative bacteria, such as E. coli, gram positive bacteria, such asany Bacillus sp. (e.g., Bacillus subtilis, Bacillus cereus), yeast hostcells (including, e.g., Pichia pastoris, Saccharomyces sp., such as S.cerevisiae and S. pombe) and Lactococcus lactis, or mammalian, fungi,plant or insect cells. The active enzyme is expressed from a variety ofconstructs in each host system. These nucleic acid expression constructscan comprise nucleotides encoding the full-length open reading frame(composed of the signal sequence, the pro-sequence, and the matureprotein coding sequence) or they can comprise a subset of these geneticelements either alone or in combination with heterologous geneticelements that serve as the signal sequence and/or the pro-sequence forthe mature open reading frame. Each of these systems can serve as acommercial production host for the expression of PLC for use in thepreviously described enzymatic oil degumming processes.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides ofthe invention, or modified nucleic acids, can be reproduced by, e.g.,amplification. The invention provides amplification primer sequencepairs for amplifying nucleic acids encoding polypeptides with aphospholipase activity. In one aspect, the primer pairs are capable ofamplifying nucleic acid sequences of the invention. One of skill in theart can design amplification primer sequence pairs for any part of orthe full length of these sequences.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a phospholipaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesphospholipases generated by amplification, e.g., polymerase chainreaction (PCR), using an amplification primer pair of the invention. Theinvention provides methods of making a phospholipase by amplification,e.g., polymerase chain reaction (PCR), using an amplification primerpair of the invention. In one aspect, the amplification primer pairamplifies a nucleic acid from a library, e.g., a gene library, such asan environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified. The skilledartisan can select and design suitable oligonucleotide amplificationprimers Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides isolated and recombinant nucleic acids comprisingsequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary nucleic acidof the invention (e.g., SEQ ID NO:5 and encoding one or more mutationsas set forth in Tables 12 to 15, as discussed in Example 3, or anenzymatically active fragment thereof, and nucleic acids encoding SEQ IDNO:6 and encoding one or more mutations as set forth in Tables 12 to 15,as discussed in Example 3, or an enzymatically active fragment thereof)over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues. The invention provides polypeptides comprising sequenceshaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to an exemplary polypeptide of theinvention. The extent of sequence identity (homology) may be determinedusing any computer program and associated parameters, including thosedescribed herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with thedefault parameters. In alternative embodiments, the sequence identifycan be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150,200, 250, 300, 350, 400 consecutive residues, or the full length of thenucleic acid or polypeptide. The extent of sequence identity (homology)may be determined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are entered into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. Default program parameters can be used, oralternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the testsequences relative to the reference sequence, based on the programparameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary sequence of theinvention are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned. Ifthe reference sequence has the requisite sequence identity to anexemplary sequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to a sequence of the invention, thatsequence is within the scope of the invention. In alternativeembodiments, subsequences ranging from about 20 to 600, about 50 to 200,and about 100 to 150 are compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequence for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search forsimilarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444, 1988, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection. Other algorithms for determininghomology or identity include, for example, in addition to a BLASTprogram (Basic Local Alignment Search Tool at the National Center forBiological Information), ALIGN, AMAS (Analysis of Multiply AlignedSequences), AMPS (Protein Multiple Sequence Alignment), ASSET (AlignedSegment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used. default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “-F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the invention, asdiscussed above, include:

-   -   “Filter for low complexity: ON    -   >Word Size: 3    -   >Matrix: Blosum62    -   >Gap Costs: Existence: 11    -   >Extension: 1”        Other default settings are: filter for low complexity OFF, word        size of 3 for protein, BLOSUM62 matrix, gap existence penalty of        −11 and a gap extension penalty of −1.

An exemplary NCBI BLAST 2.2.2 program setting is set forth in Example 1,below. Note that the “-W” option defaults to 0. This means that, if notset, the word size defaults to 3 for proteins and 11 for nucleotides.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to an exemplary sequence ofthe invention, e.g., a sequence as set forth in SEQ ID NO:5 and havingone or more mutations as set forth in Tables 12 to 15, as described inExample 3, below, or a nucleic acid that encodes a polypeptidecomprising a sequence as set forth in SEQ ID NO:6 and encoding one ormore mutations as set forth in Tables 12 to 15, or an enzymaticallyactive fragment thereof.

The stringent conditions can be highly stringent conditions, mediumstringent conditions, low stringent conditions, including the high andreduced stringency conditions described herein. In alternativeembodiments, nucleic acids of the invention as defined by their abilityto hybridize under stringent conditions can be between about fiveresidues and the full length of the molecule, e.g., an exemplary nucleicacid of the invention. For example, they can be at least 5, 10, 15, 20,25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300,350, 400 or more residues in length. Nucleic acids shorter than fulllength are also included. These nucleic acids are useful as, e.g.,hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA(single or double stranded), antisense or sequences encoding antibodybinding peptides (epitopes), motifs, active sites, binding domains,regulatory domains and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C. Alternatively, nucleic acids of the invention aredefined by their ability to hybridize under high stringency comprisingconditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and arepetitive sequence blocking nucleic acid, such as cot-1 or salmon spermDNA (e.g., 200 ug/ml sheared and denatured salmon sperm DNA). In oneaspect, nucleic acids of the invention are defined by their ability tohybridize under reduced stringency conditions comprising 35% formamideat a reduced temperature of 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionscan be used to practice the invention and are well known in the art.Hybridization conditions are discussed further, below.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes for identifying and/orisolating a nucleic acid encoding a polypeptide having a phospholipaseactivity. In one aspect, the probe comprises or consists of a nucleicacid of the invention, e.g., a nucleic acid having a sequence as setforth in SEQ ID NO:5 and having one or more base changes (mutations) asset forth in Tables 12 to 15, as described in Example 3, below, or anucleic acid that encodes a polypeptide comprising a sequence as setforth in SEQ ID NO:6 and encoding one or more amino acid residue changes(mutations) as set forth in Tables 12 to 15, or an enzymatically activefragment thereof. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, or 150, or more, or about 10 to 50, about 20 to 60 about 30 to 70,consecutive bases of a nucleic acid sequence of the invention.

The probes identify a nucleic acid by binding or hybridization. Inalternative embodiments, hybridization comprises the process by which anucleic acid strand joins with a complementary strand through basepairing. Hybridization reactions can be sensitive and selective so thata particular sequence of interest can be identified even in samples inwhich it is present at low concentrations. Suitably stringent conditionscan be defined by, for example, the concentrations of salt or formamidein the prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. For example,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature, altering the time of hybridization, as described in detail,below. In alternative aspects, nucleic acids of the invention aredefined by their ability to hybridize under various stringencyconditions (e.g., high, medium, and low), as set forth herein.

The probes can be used in arrays of the invention, see discussion below,including, e.g., capillary arrays. The probes of the invention can alsobe used to isolate and/or identify other phospholipase-encoding nucleicacids or polypeptides having a phospholipase activity.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention. In such procedures, a biologicalsample potentially harboring the organism from which the nucleic acidwas isolated is obtained and nucleic acids are obtained from the sample.The nucleic acids are contacted with the probe under conditions whichpermit the probe to specifically hybridize to any complementarysequences present in the sample. Where necessary, conditions whichpermit the probe to specifically hybridize to complementary sequencesmay be determined by placing the probe in contact with complementarysequences from samples known to contain the complementary sequence, aswell as control sequences which do not contain the complementarysequence. Hybridization conditions, such as the salt concentration ofthe hybridization buffer, the formamide concentration of thehybridization buffer, or the hybridization temperature, may be varied toidentify conditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. In alternative embodiments, a nucleic acid isimmobilized, for example, on a filter. Hybridization may be carried outunder conditions of low stringency, moderate stringency or highstringency. As an example of nucleic acid hybridization, a polymermembrane containing immobilized denatured nucleic acids is firstprehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 MNaCl, 50 mM NaH₂PO4, pH 7.0, 5.0 mM Na₂EDTA, 0.5% SDS, 10×Denhardt's,and 0.5 mg/ml polyriboadenylic acid. Approximately 2×10⁷ cpm (specificactivity 4 to 9×10⁸ cpm/ug) of ³²P end-labeled oligonucleotide probe canbe added to the solution. In alternative embodiments, after about 12 to16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane canbe exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is thelength of the probe. Prehybridization may be carried out in 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denaturedfragmented salmon sperm DNA, 50% formamide. Formulas for SSC andDenhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes that can be used topractice this invention are: 2×SSC, 0.1% SDS at room temperature for 15minutes (low stringency); 0.1×SSC, 0.5% SDS at room temperature for 30minutes to 1 hour (moderate stringency); 0.1×SSC, 0.5% SDS for 15 to 30minutes at between the hybridization temperature and 68° C. (highstringency); and 0.15M NaCl for 15 minutes at 72° C. (very highstringency). A final low stringency wash can be conducted in 0.1×SSC atroom temperature. The examples above are merely illustrative of one setof conditions that can be used to practice the invention, e.g., to washfilters or arrays. One of skill in the art would know that there arenumerous recipes for different stringency washes, all of which can beused to practice the invention.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

In alternative embodiments, the hybridization is carried out in buffers,such as 6×SSC, containing formamide at a temperature of 42° C. In thiscase, the concentration of formamide in the hybridization buffer may bereduced in 5% increments from 50% to 0% to identify clones havingdecreasing levels of homology to the probe. Following hybridization, thefilter may be washed with 6×SSC, 0.5% SDS at 50° C. In alternativeembodiments, “moderate” conditions are above 25% formamide and “low”conditions are below 25% formamide. In alternative embodiments,“moderate” hybridization conditions is when the above hybridization isconducted at 30% formamide. In alternative embodiments, “low stringency”hybridization conditions is when the above hybridization is conducted at10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 99%, at least 98%, at least97%, at least 96%, at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 65%, at least 60%, at least55%, or at least 50% homology to a nucleic acid sequence of theinvention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 250, 300, 350, 400, or 500 consecutive bases thereof, andthe sequences complementary thereto. Homology may be measured using analignment algorithm, as discussed herein. For example, the homologouspolynucleotides may have a coding sequence which is a naturallyoccurring allelic variant of one of the coding sequences describedherein. Such allelic variants may have a substitution, deletion oraddition of one or more nucleotides when compared to nucleic acids ofthe invention.

Additionally, the probes and methods of the invention may be used toisolate nucleic acids which encode polypeptides having at least about99%, at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least50% sequence identity (homology) to a polypeptide of the inventioncomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof as determined using a sequence alignmentalgorithm (e.g., such as the FASTA version 3.0t78 algorithm with thedefault parameters, or a BLAST 2.2.2 program with exemplary settings asset forth herein).

Inhibiting Expression of Phospholipases

The invention further provides nucleic acids complementary to (e.g.,antisense sequences to) the nucleic acids of the invention, e.g.,phospholipase-encoding nucleic acids. Antisense sequences are capable ofinhibiting the transport, splicing or transcription ofphospholipase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA (mRNA, a transcript). Thetranscription or function of targeted nucleic acid can be inhibited, forexample, by hybridization and/or cleavage. One particularly useful setof inhibitors provided by the present invention includesoligonucleotides which are able to either bind phospholipase gene ormessage, in either case preventing or inhibiting the production orfunction of phospholipase enzyme. The association can be though sequencespecific hybridization. Another useful class of inhibitors includesoligonucleotides which cause inactivation or cleavage of phospholipasemessage. The oligonucleotide can have enzyme activity which causes suchcleavage, such as ribozymes. The oligonucleotide can be chemicallymodified or conjugated to an enzyme or composition capable of cleavingthe complementary nucleic acid. One may screen a pool of many differentsuch oligonucleotides for those with the desired activity.

Inhibition of phospholipase expression can have a variety of industrialapplications. For example, inhibition of phospholipase expression canslow or prevent spoilage. Spoilage can occur when lipids orpolypeptides, e.g., structural lipids or polypeptides, are enzymaticallydegraded. This can lead to the deterioration, or rot, of fruits andvegetables. In one aspect, use of compositions of the invention thatinhibit the expression and/or activity of phospholipase, e.g.,antibodies, antisense oligonucleotides, ribozymes and RNAi, are used toslow or prevent spoilage. Thus, in one aspect, the invention providesmethods and compositions comprising application onto a plant or plantproduct (e.g., a fruit, seed, root, leaf, etc.) antibodies, antisenseoligonucleotides, ribozymes and RNAi of the invention to slow or preventspoilage. These compositions also can be expressed by the plant (e.g., atransgenic plant) or another organism (e.g., a bacterium or othermicroorganism transformed with a phospholipase gene of the invention).

The compositions of the invention for the inhibition of phospholipaseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingphospholipase message which can inhibit phospholipase activity bytargeting mRNA. Strategies for designing antisense oligonucleotides arewell described in the scientific and patent literature, and the skilledartisan can design such phospholipase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl. Pharmacol. 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensephospholipase sequences of the invention (see, e.g., Gold (1995) J. ofBiol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides for with ribozymes capable of bindingphospholipase message which can inhibit phospholipase enzyme activity bytargeting mRNA. Strategies for designing ribozymes and selecting thephospholipase-specific antisense sequence for targeting are welldescribed in the scientific and patent literature, and the skilledartisan can design such ribozymes using the novel reagents of theinvention. Ribozymes act by binding to a target RNA through the targetRNA binding portion of a ribozyme which is held in close proximity to anenzymatic portion of the RNA that cleaves the target RNA. Thus, theribozyme recognizes and binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocleave and inactivate the target RNA. Cleavage of a target RNA in such amanner will destroy its ability to direct synthesis of an encodedprotein if the cleavage occurs in the coding sequence. After a ribozymehas bound and cleaved its RNA target, it is typically released from thatRNA and so can bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif,but may also be formed in the motif of a hairpin, hepatitis delta virus,group I intron or RNaseP-like RNA (in association with an RNA guidesequence). Examples of such hammerhead motifs are described by Rossi(1992) Aids Research and Human Retroviruses 8:183; hairpin motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a phospholipase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a phospholipase gene. Inone aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25or more duplex nucleotides in length. While the invention is not limitedby any particular mechanism of action, the RNAi can enter a cell andcause the degradation of a single-stranded RNA (ssRNA) of similar oridentical sequences, including endogenous mRNAs. When a cell is exposedto double-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a phospholipase enzyme. Inalternative embodiment, the invention provides methods for modifying anenzyme of the invention, e.g., by mutation of its coding sequence byrandom or stochastic methods, or, non-stochastic, or “directedevolution,” such as Gene Site Saturation Mutagenesis™ (GSSM), to alterthe enzymes pH range of activity or range of optimal activity,temperature range of activity or range of optimal activity, specificity,activity (kinetics); the enzyme's use of glycosylation, phosphorylationor metals (e.g., Ca, Mg, Zn, Fe, Na), e.g., to impact pH/temperaturestability. The invention provides methods for modifying an enzyme of theinvention, e.g., by mutation of its coding sequence, e.g., by GSSM, toincrease its resistance to protease activity. The invention providesmethods for modifying an enzyme of the invention, e.g., by mutation ofits coding sequence, e.g., by GSSM, to modify the enzyme's use of metalchelators specific for Ca, Mg, Na that would not chelate Zn. Theinvention provides methods for modifying an enzyme of the invention,e.g., by mutation of its coding sequence, e.g., by GSSM, that would havea desired combination of activities, e.g., PI, PA and PC/PE specificPLCs.

In one embodiment, “Gene Site Saturation Mutagenesis” (GSSM) or “GSSM”comprises a method that uses degenerate oligonucleotide primers tointroduce point mutations into a polynucleotide, as described in detail,below. In one embodiment, “optimized directed evolution system” or“optimized directed evolution” comprises a method for reassemblingfragments of related nucleic acid sequences, e.g., related genes, andexplained in detail, below. In one embodiment, “synthetic ligationreassembly” or “SLR” comprises a method of ligating oligonucleotidefragments in a non-stochastic fashion, and explained in detail, below.

In alternative embodiments, the invention provides “variants” ofexemplary nucleic acids and polypeptides of the invention, includinge.g., SEQ ID NO:8, encoded e.g., by SEQ ID NO:7, SEQ ID NO:8 or SEQ IDNO:9. In alternative embodiments variants of polynucleotides orpolypeptides of the invention are nucleic acids or polypeptides thathave been modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a phospholipase. Variants can be produced by any number ofmeans included methods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, GSSM and any combination thereof.Techniques for producing variant phospholipases having activity at a pHor temperature, for example, that is different from a wild-typephospholipase, are included herein.

These methods can be repeated or used in various combinations togenerate phospholipase enzymes having an altered or different activityor an altered or different stability from that of a phospholipaseencoded by the template nucleic acid. These methods also can be repeatedor used in various combinations, e.g., to generate variations ingene/message expression, message translation or message stability. Inanother aspect, the genetic composition of a cell is altered by, e.g.,modification of a homologous gene ex vivo, followed by its reinsertioninto the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods.

Methods for random mutation of genes are well known in the art, see,e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be used torandomly mutate a gene. Mutagens include, e.g., ultraviolet light orgamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid,photoactivated psoralens, alone or in combination, to induce DNA breaksamenable to repair by recombination. Other chemical mutagens include,for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine orformic acid. Other mutagens are analogues of nucleotide precursors,e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include pointmismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887),mutagenesis using repair-deficient host strains (Carter et al. (1985)“Improved oligonucleotide site-directed mutagenesis using M13 vectors”Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Certain U.S. applications provide additional details regarding variousdiversity generating methods, including “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No. 09/407,800);“EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCERECOMBINATION” by del Cardayre et al., filed Jul. 15, 1998 (U.S. Ser.No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922);“OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al.,filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and “OLIGONUCLEOTIDEMEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18,2000 (PCT/US00/01203); “USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESISFOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser.No. 09/408,393); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES& POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al.,filed Jan. 18, 2000, (PCT/US00/01202) and, e.g. “METHODS FOR MAKINGCHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIREDCHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.09/618,579); “METHODS OF POPULATING DATA STRUCTURES FOR USE INEVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000(PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATEDRECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter, filedSep. 6, 2000 (U.S. Ser. No. 09/656,549).

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (e.g., GSSM), synthetic ligation reassembly(SLR), or a combination thereof are used to modify the nucleic acids ofthe invention to generate phospholipases with new or altered properties(e.g., activity under highly acidic or alkaline conditions, hightemperatures, and the like). Polypeptides encoded by the modifiednucleic acids can be screened for an activity before testing for aphospholipase or other activity. Any testing modality or protocol can beused, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos.6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect of the invention, non-stochastic gene modification, a“directed evolution process,” is used to generate phospholipases withnew or altered properties. Variations of this method have been termed“gene site mutagenesis,” “site-saturation mutagenesis,” “Gene SiteSaturation Mutagenesis” or simply “GSSM.” It can be used in combinationwith other mutagenization processes. See, e.g., U.S. Pat. Nos.6,171,820; 6,238,884. In one aspect, GSSM comprises providing a templatepolynucleotide and a plurality of oligonucleotides, wherein eacholigonucleotide comprises a sequence homologous to the templatepolynucleotide, thereby targeting a specific sequence of the templatepolynucleotide, and a sequence that is a variant of the homologous gene;generating progeny polynucleotides comprising non-stochastic sequencevariations by replicating the template polynucleotide with theoligonucleotides, thereby generating polynucleotides comprisinghomologous gene sequence variations.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, so as togenerate a set of progeny polypeptides in which a full range of singleamino acid substitutions is represented at each amino acid position,e.g., an amino acid residue in an enzyme active site or ligand bindingsite targeted to be modified. These oligonucleotides can comprise acontiguous first homologous sequence, a degenerate N,N,G/T sequence,and, optionally, a second homologous sequence. The downstream progenytranslational products from the use of such oligonucleotides include allpossible amino acid changes at each amino acid site along thepolypeptide, because the degeneracy of the N,N,G/T sequence includescodons for all 20 amino acids. In one aspect, one such degenerateoligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) isused for subjecting each original codon in a parental polynucleotidetemplate to a full range of codon substitutions. In another aspect, atleast two degenerate cassettes are used—either in the sameoligonucleotide or not, for subjecting at least two original codons in aparental polynucleotide template to a full range of codon substitutions.For example, more than one N,N,G/T sequence can be contained in oneoligonucleotide to introduce amino acid mutations at more than one site.This plurality of N,N,G/T sequences can be directly contiguous, orseparated by one or more additional nucleotide sequence(s). In anotheraspect, oligonucleotides serviceable for introducing additions anddeletions can be used either alone or in combination with the codonscontaining an N,N,G/T sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,phospholipase) molecules such that all 20 natural amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide (other aspectsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g. cloned into asuitable host, e.g., E. coli host, using, e.g., an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedphospholipase activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined -6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate phospholipases with new or altered properties. SLRis a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. patent application Ser. No.09/332,835 entitled “Synthetic Ligation Reassembly in DirectedEvolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). Inone aspect, SLR comprises the following steps: (a) providing a templatepolynucleotide, wherein the template polynucleotide comprises sequenceencoding a homologous gene; (b) providing a plurality of building blockpolynucleotides, wherein the building block polynucleotides are designedto cross-over reassemble with the template polynucleotide at apredetermined sequence, and a building block polynucleotide comprises asequence that is a variant of the homologous gene and a sequencehomologous to the template polynucleotide flanking the variant sequence;(c) combining a building block polynucleotide with a templatepolynucleotide such that the building block polynucleotide cross-overreassembles with the template polynucleotide to generate polynucleotidescomprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled.

In one aspect of this method, the sequences of a plurality of parentalnucleic acid templates are aligned in order to select one or moredemarcation points. The demarcation points can be located at an area ofhomology, and are comprised of one or more nucleotides. Thesedemarcation points are preferably shared by at least two of theprogenitor templates. The demarcation points can thereby be used todelineate the boundaries of oligonucleotide building blocks to begenerated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in anotherembodiment, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic) asdescribed above. Because of the non-stochastic nature of this invention,the possibility of unwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecularly homologousdemarcation points and thus to allow an increased number of couplings tobe achieved among the building blocks, which in turn allows a greaternumber of progeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate phospholipases withnew or altered properties. Optimized directed evolution is directed tothe use of repeated cycles of reductive reassortment, recombination andselection that allow for the directed molecular evolution of nucleicacids through recombination. Optimized directed evolution allowsgeneration of a large population of evolved chimeric sequences, whereinthe generated population is significantly enriched for sequences thathave a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found in U.S. Ser. No.09/332,835. The number of oligonucleotides generated for each parentalvariant bears a relationship to the total number of resulting crossoversin the chimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding an polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that aoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. One can calculate such a probability density function,and thus enrich the chimeric progeny population for a predeterminednumber of crossover events resulting from a particular ligationreaction. Moreover, a target number of crossover events can bepredetermined, and the system then programmed to calculate the startingquantities of each parental oligonucleotide during each step in theligation reaction to result in a probability density function thatcenters on the predetermined number of crossover events.

Determining Crossover Events

Embodiments of the invention include a system and software that receivea desired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB® (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example a nucleic acid (or, the nucleic acid) responsiblefor an altered phospholipase phenotype is identified, re-isolated, againmodified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including phospholipase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new phospholipasephenotype), it can be removed as a variable by synthesizing largerparental oligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,phospholipase enzymes, and the like. In vivo shuffling can be performedutilizing the natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides methods of making sequence variants of thenucleic acid and phospholipase sequences of the invention or isolatingphospholipase enzyme, e.g., phospholipase, sequence variants using thenucleic acids and polypeptides of the invention. In one aspect, theinvention provides for variants of a phospholipase gene of theinvention, which can be altered by any means, including, e.g., random orstochastic methods, or, non-stochastic, or “directed evolution,”methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In someembodiments, random mutations in a sequence of interest are generated bypropagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some embodiments, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250.

The invention also provides variants of polypeptides of the inventioncomprising sequences in which one or more of the amino acid residues(e.g., of an exemplary polypeptide of the invention) are substitutedwith a conserved or non-conserved amino acid residue (e.g., a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code. Conservative substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Thus, polypeptides of the inventioninclude those with conservative substitutions of sequences of theinvention, including but not limited to the following replacements:replacements of an aliphatic amino acid such as Alanine, Valine, Leucineand Isoleucine with another aliphatic amino acid; replacement of aSerine with a Threonine or vice versa; replacement of an acidic residuesuch as Aspartic acid and Glutamic acid with another acidic residue;replacement of a residue bearing an amide group, such as Asparagine andGlutamine, with another residue bearing an amide group; exchange of abasic residue such as Lysine and Arginine with another basic residue;and replacement of an aromatic residue such as Phenylalanine, Tyrosinewith another aromatic residue. Other variants are those in which one ormore of the amino acid residues of the polypeptides of the inventionincludes a substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., a phospholipase activity,as described herein. In other aspects, the variant, fragment,derivative, or analog includes a proprotein, such that the variant,fragment, derivative, or analog can be activated by cleavage of theproprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying phospholipase-encodingnucleic acids to modify codon usage. In one aspect, the inventionprovides methods for modifying codons in a nucleic acid encoding aphospholipase to increase or decrease its expression in a host cell. Theinvention also provides nucleic acids encoding a phospholipase modifiedto increase its expression in a host cell, phospholipase enzymes somodified, and methods of making the modified phospholipase enzymes. Themethod comprises identifying a “non-preferred” or a “less preferred”codon in phospholipase-encoding nucleic acid and replacing one or moreof these non-preferred or less preferred codons with a “preferred codon”encoding the same amino acid as the replaced codon and at least onenon-preferred or less preferred codon in the nucleic acid has beenreplaced by a preferred codon encoding the same amino acid. A preferredcodon is a codon over-represented in coding sequences in genes in thehost cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as any Bacillus (e.g., B. cereus) orStreptomyces, Lactobacillus gasseri, Lactococcus lactis, Lactococcuscremoris, Bacillus subtilis. Exemplary host cells also includeeukaryotic organisms, e.g., various yeast, such as Saccharomyces sp.,including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillusniger, and mammalian cells and cell lines and insect cells and celllines. Thus, the invention also includes nucleic acids and polypeptidesoptimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding a phospholipaseisolated from a bacterial cell are modified such that the nucleic acidis optimally expressed in a bacterial cell different from the bacteriafrom which the phospholipase was derived, a yeast, a fungi, a plantcell, an insect cell or a mammalian cell. Methods for optimizing codonsare well known in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca(2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif.12:185-188; Narum (2001) Infect. Immun. 69:7250-7253. See also Narum(2001) Infect. Immun. 69:7250-7253, describing optimizing codons inmouse systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,describing optimizing codons in yeast; Feng (2000) Biochemistry39:15399-15409, describing optimizing codons in E. coli; Humphreys(2000) Protein Expr. Purif. 20:252-264, describing optimizing codonusage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide, an expression cassette or vector or a transfectedor transformed cell of the invention. The transgenic non-human animalscan be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice,comprising the nucleic acids of the invention. These animals can beused, e.g., as in vivo models to study phospholipase activity, or, asmodels to screen for modulators of phospholipase activity in vivo. Thecoding sequences for the polypeptides to be expressed in the transgenicnon-human animals can be designed to be constitutive, or, under thecontrol of tissue-specific, developmental-specific or inducibletranscriptional regulatory factors. Transgenic non-human animals can bedesigned and generated using any method known in the art; see, e.g.,U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usingtransformed cells and eggs and transgenic mice, rats, rabbits, sheep,pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods231:147-157, describing the production of recombinant proteins in themilk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol.17:456-461, demonstrating the production of transgenic goats. U.S. Pat.No. 6,211,428, describes making and using transgenic non-human mammalswhich express in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice whose cells express proteins related to thepathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express or to be unable to express aphospholipase.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a phospholipase), an expression cassette orvector or a transfected or transformed cell of the invention. Theinvention also provides plant products, e.g., oils, seeds, leaves,extracts and the like, comprising a nucleic acid and/or a polypeptide(e.g., a phospholipase) of the invention. The transgenic plant can bedicotyledonous (a dicot) or monocotyledonous (a monocot). The inventionalso provides methods of making and using these transgenic plants andseeds. The transgenic plant or plant cell expressing a polypeptide ofthe invention may be constructed in accordance with any method known inthe art. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's phospholipase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on oil-seed containing plants, such asrice, soybeans, rapeseed, sunflower seeds, sesame and peanuts. Nucleicacids of the invention can be used to manipulate metabolic pathways of aplant in order to optimize or alter host's expression of phospholipase.The can change phospholipase activity in a plant. Alternatively, aphospholipase of the invention can be used in production of a transgenicplant to produce a compound not naturally produced by that plant. Thiscan lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. For example, aconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation and microinjection ofplant cell protoplasts, or the constructs can be introduced directly toplant tissue using ballistic methods, such as DNA particle bombardment.For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with nucleicacids, e.g., an expression construct. Although plant regeneration fromprotoplasts is not easy with cereals, plant regeneration is possible inlegumes using somatic embryogenesis from protoplast derived callus.Organized tissues can be transformed with naked DNA using gene guntechnique, where DNA is coated on tungsten microprojectiles, shot1/100th the size of cells, which carry the DNA deep into cells andorganelles. Transformed tissue is then induced to regenerate, usually bysomatic embryogenesis. This technique has been successful in severalcereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., aphospholipase) of the invention. The desired effects can be passed tofuture plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants (e.g., as transgenic plants), such as oil-seedcontaining plants, e.g., rice, soybeans, rapeseed, sunflower seeds,sesame and peanuts. The nucleic acids of the invention can be expressedin plants which contain fiber cells, including, e.g., cotton, silkcotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., a phospholipase orantibody) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas 1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

The invention provides isolated, synthetic or recombinant polypeptideshaving a sequence identity (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity) to an exemplarysequence of the invention, e.g., SEQ ID NO:6 having one or more sequencechanges (e.g., mutations) as set forth in Tables 12 to 15, as discussedin Example 3, below, or an enzymatically active fragment thereof.

As discussed above, the identity can be over the full length of thepolypeptide, or, the identity can be over a subsequence thereof, e.g., aregion of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700 or more residues. Polypeptides ofthe invention can also be shorter than the full length of exemplarypolypeptides. In alternative embodiment, the invention providespolypeptides (peptides, fragments) ranging in size between about 5 andthe full length of a polypeptide, e.g., an enzyme, such as aphospholipase, e.g., phospholipase; exemplary sizes being of about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400 or more residues, e.g.,contiguous residues of the exemplary phospholipases. Peptides of theinvention can be useful as, e.g., labeling probes, antigens, toleragens,motifs, phospholipase active sites, binding domains, regulatory domains,and the like.

In one aspect, the invention provides polypeptides having sequences asset forth in SEQ ID NO:6 comprising (and having) one or more amino acidresidue changes (e.g., mutations) as set forth in Tables 12 to 15, andsubsequences thereof, e.g., their active sites (“catalytic domains”)having a phospholipase activity, e.g., a phospholipase C (PLC) activity,e.g., a PI-PLC activity. In one aspect, the polypeptide has aphospholipase activity but lacks neutral oil (triglyceride) hydrolysisactivity. For example, in one aspect, the polypeptide has aphospholipase activity but lacks any activity that affects a neutral oil(triglyceride) fraction. In one aspect, the invention provides adegumming process comprising use of a polypeptide of the inventionhaving a phospholipase activity, but not a lipase activity.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules.

The terms “polypeptide” and “protein” as used herein, refer to aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres, and may contain modified amino acids other thanthe 20 gene-encoded amino acids. The term “polypeptide” also includespeptides and polypeptide fragments, motifs and the like. The term alsoincludes glycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, an isolated material or composition can also be a“purified” composition, i.e., it does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library can be conventionally purified toelectrophoretic homogeneity. In alternative aspects, the inventionprovides nucleic acids which have been purified from genomic DNA or fromother sequences in a library or other environment by at least one, two,three, four, five or more orders of magnitude.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has aphospholipase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-di-isopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH2- for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenyl-phenylalanine; K- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole-(alkyl)alanines;and, D- or L-alkylainines, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromaticrings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl,pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′-N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R— or S— form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

Phospholipase Enzymes

The invention provides polypeptides having a phospholipase activity,nucleic acids encoding them, antibodies that bind them, peptidesrepresenting the enzyme's antigenic sites (epitopes) and active sites,regulatory and binding domains, and methods for making and using them.In one aspect, polypeptides of the invention have a phospholipaseactivity, or any combination of phospholipase activities, as describedherein (e.g., a phosphatidylinositol-specific phospholipase C (PI-PLC)enzyme activity, etc.). In alternative aspects, the phospholipases ofthe invention have activities that have been modified from those of theexemplary phospholipases described herein.

As used herein, the term “phospholipase” encompasses enzymes having anyphospholipase activity, for example, cleaving a glycerolphosphate esterlinkage (catalyzing hydrolysis of a glycerolphosphate ester linkage),e.g., in an oil, such as a crude oil or a vegetable oil. Thephospholipase activity of the invention can generate a water extractablephosphorylated base and a diglyceride. The term “a phospholipaseactivity” hydrolysis of glycerolphosphate ester linkages at hightemperatures, low temperatures, alkaline pHs and at acidic pHs, cleavinga glycerolphosphate ester to generate a water extractable phosphorylatedbase and a diglyceride, cutting ester bonds of glycerin and phosphoricacid in phospholipids, and other activities, such as the ability to bindto and hydrolyze a substrate, such as an oil, e.g. a crude oil or avegetable oil, substrate also including plant and animalphosphatidylcholines, phosphatidylethanolamines, phosphatidylserines andsphingomyelins. The phospholipase activity can comprise a phospholipaseC (PLC) activity; a PI-PLC activity, a phospholipase A (PLA) activity,such as a phospholipase A1 or phospholipase A2 activity; a phospholipaseB (PLB) activity, such as a phospholipase B1 or phospholipase B2activity, including lysophospholipase (LPL) activity and/orlysophospholipase-transacylase (LPTA) activity; a phospholipase D (PLD)activity, such as a phospholipase D1 or a phospholipase D2 activity;and/or a patatin activity or any combination thereof. The phospholipaseactivity can comprise hydrolysis of a glycoprotein, e.g., as aglycoprotein found in a potato tuber or any plant of the genus Solanum,e.g., Solanum tuberosum. In alternative embodiments, the phospholipaseactivity can comprise a patatin enzymatic activity, such as a patatinesterase activity (see, e.g., Jimenez (2002) Biotechnol. Prog.18:635-640). In certain embodiments, the phospholipase activity cancomprise a lipid acyl hydrolase (LAH) activity.

In alternative embodiments, the PLC phospholipases of the inventionutilize (e.g., catalyze hydrolysis of) a variety of phospholipidsubstrates including phosphatidylcholine (PC), phosphatidylethanolamine(PE), phosphatidylserine (PS), phosphatidylinositol (PI), and/orphosphatidic acid (PA) or a combination thereof. In addition, theseenzymes can have varying degrees of activity on the lysophospholipidforms of these phospholipids. In various aspects, PLC enzymes of theinvention may show a preference for phosphatidylcholine andphosphatidylethanolamine as substrates.

In alternative embodiments, the phosphatidylinositol PLC phospholipasesof the invention utilize a variety of phospholipid substrates includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, and phosphatidic acid, or a combination thereof.In alternative embodiments, these enzymes can have varying degrees ofactivity on the lysophospholipid forms of these phospholipids. Invarious aspects, phosphatidylinositol PLC enzymes of the invention mayshow a preference for phosphatidylinositol as a substrate.

In alternative embodiments, the phospholipase activity can comprisebeing specific for one or more specific substrates, e.g., an enzyme ofthe invention can have a specificity of action for PE and PC; PE an PI;PE and PS; PS and PC; PS and PI; PI and PC; PS, PI and PC; PE, PI andPC; PC, PE and PS; PE, PS and PI; or, PE, PS, PI and PC, or anycombination thereof.

In alternative embodiments, a phospholipase of the invention can havemultifunctional activity, e.g., a combination of one or more of theenzyme activities described herein. For example, in one aspect, apolypeptide of the invention is enzymatically active, but lacks a lipaseactivity or lacks any enzymatic activity that affects a neutral oil(triglyceride) fraction. It may be desirable to use such a polypeptidein a particular process, e.g., in a degumming process where it isimportant that the neutral oil fraction not be harmed (diminished,degraded, e.g., hydrolyzed). Thus, in one aspect, the invention providesa degumming process comprising use of a polypeptide of the inventionhaving a phospholipase activity, but not a lipase activity.

In alternative embodiments, polypeptides of the invention having patatinenzyme activity can utilize a variety of phospholipid substratesincluding phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and phosphatidic acid, or acombination thereof. In addition, these enzymes can have varying degreesof activity on the lysophospholipid forms of these phospholipids. Invarious aspects, patatins of the invention are based on a conservationof amino acid sequence similarity. In various aspects, these enzymesdisplay a diverse set of biochemical properties and may performreactions characteristic of PLA1, PLA2, PLC, or PLD enzyme classes.

In alternative embodiments, polypeptides of the invention having PLDphospholipases of the invention can utilize a variety of phospholipidsubstrates including phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and phosphatidic acid, or acombination thereof. In addition, these enzymes can have varying degreesof activity on the lysophospholipid forms of these phospholipids. In oneaspect, these enzymes are useful for carrying out transesterificationreactions to produce structured phospholipids.

In alternative embodiments, polypeptides of the invention have anactivity comprising cleavage of a glycerolphosphate ester linkage, theability to hydrolyze phosphate ester bonds, including patatin, lipidacyl hydrolase (LAH), phospholipase A, B, C and/or phospholipase Dactivity, or any combination thereof.

As used herein, 1 enzyme unit is the quantity of an enzyme needed tocause a reaction to process 1 micromole of substance per minute underspecified conditions.

In alternative embodiments, the invention provides polypeptides with andwithout signal sequences, and the signal sequences themselves (e.g.,isolated signal sequence peptides). The invention includes fragments orsubsequences of enzymes of the invention, e.g., peptides or polypeptidescomprising or consisting of catalytic domains (“active sites”), bindingsites, regulatory domains, epitopes, signal sequences, prepro domains,and the like. The invention also includes immobilized phospholipases,anti-phospholipase antibodies and fragments thereof. The inventionincludes heterocomplexes, e.g., fusion proteins, heterodimers, etc.,comprising the phospholipases of the invention. Determining peptidesrepresenting the enzyme's antigenic sites (epitopes), active sites,binding sites, signal sequences, and the like can be done by routinescreening protocols.

These enzymes and processes of the invention can be used to achieve amore complete degumming of high phosphorus oils, in particular, rice,soybean, corn, canola, and sunflower oils. For example, in one aspect,upon cleavage by PI-PLC, phosphatidylinositol is converted todiacylglycerol and phosphoinositol. The diacylglycerol partitions to theaqueous phase (improving oil yield) and the phosphoinositol partitionsto the aqueous phase where it is removed as a component of the heavyphase during centrifugation. An enzyme of the invention, e.g., a PI-PLCof the invention, can be incorporated into either a chemical or physicaloil refining process.

In alternative aspects, enzymes of the invention havephosphatidylinositol-specific phospholipase C (PI-PLC) activity,phosphatidylcholine-specific phospholipase C activity, phosphatidic acidphosphatase activity, phospholipase A activity and/or patatin-relatedphospholipase activity. These enzymes can be used alone or incombination each other or with other enzymes of the invention, or otherenzymes. In one aspect, the invention provides methods wherein theseenzymes (including phosphatidylinositol-specific phospholipase C(PIPLC), phosphatidylcholine-specific phospholipase C, and/orphospholipase D (in conjunction with a phosphatase), phosphatidic acidphosphatase, phospholipase A, patatin-related phospholipases of theinvention) are used alone or in combination in the degumming of oils,e.g., vegetable oils, e.g., high phosphorus oils, such as soybean, corn,canola, rice bran and sunflower oils. These enzymes and processes of theinvention can be used to achieve a more complete degumming of highphosphorus oils, in particular, soybean, corn, canola, rice bran andsunflower oils. Upon cleavage by PI-PLC, phosphatidylinositol isconverted to diacylglycerol and phosphoinositol. The diacylglycerolpartitions to the aqueous phase (improving oil yield) and thephosphoinositol partitions to the aqueous phase where it is removed as acomponent of the heavy phase during centrifugation. An enzyme of theinvention, e.g., a PI-PLC of the invention, can be incorporated intoeither a chemical or physical oil refining process.

In one aspect, the invention provides compositions, e.g., solutions,comprising sodium citrate at neutral pH to hydrate non-hydratables. Forexample, the invention provides sodium citrate solutions in a pH rangeof between about 4 to 9, or, 5 to 8, or, 6 to 7, that can be used tohydrate non-hydratable phospholipids (including enzymes of theinvention) in high phosphorus oils. In one aspect, the hydration ofnon-hydratable phospholipids is by chelating the calcium and magnesiumassociated with the phospholipids, thereby allowing the formerlyinsoluble phospholipid salts to more readily partition in the aqueousphase. In one aspect, once phospholipids move to the water/oil interfaceor into the aqueous phase, a phospholipase of the invention (e.g., aphospholipase-specific phosphohydrolase of the invention), or anotherphospholipase, will convert the phospholipid to diacylglycerol and aphosphate-ester. In one aspect, calcium and magnesium metal content arelowered upon addition of acid and caustic (see discussion on causticprocesses).

The enzymes of the invention are highly selective catalysts. As withother enzymes, they catalyze reactions with exquisite stereo-, regio-,and chemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, the enzymes of the invention are remarkablyversatile. They can be tailored to function in organic solvents, operateat extreme pHs (for example, high pHs and low pHs) extreme temperatures(for example, high temperatures and low temperatures), extreme salinitylevels (for example, high salinity and low salinity), and catalyzereactions with compounds that are structurally unrelated to theirnatural, physiological substrates. Enzymes of the invention can bedesigned to be reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Enzymes of the invention can also be designed to be highlyenantio- and regio-selective. The high degree of functional groupspecificity exhibited by these enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes of the invention can also be designed to catalyze many diversereactions unrelated to their native physiological function in nature.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound. The present invention usesselected biocatalysts, i.e., the enzymes of the invention, and reactionconditions that are specific for functional groups that are present inmany starting compounds. Each biocatalyst is specific for one functionalgroup, or several related functional groups, and can react with manystarting compounds containing this functional group. The biocatalyticreactions produce a population of derivatives from a single startingcompound. These derivatives can be subjected to another round ofbiocatalytic reactions to produce a second population of derivativecompounds. Thousands of variations of the original compound can beproduced with each iteration of biocatalytic derivatization.

The invention provides methods for identifying a single active PLCenzyme within a library, where the library is characterized by theseries of biocatalytic reactions used to produce it, a so-called“biosynthetic history”. One embodiment comprises screening the libraryfor biological activities and tracing the biosynthetic historyidentifies the specific reaction sequence producing the active compound.The reaction sequence can be repeated and the structure of thesynthesized compound determined. In this embodiment, for this mode ofidentification, an immobilization technology is not required; compoundscan be synthesized and tested free in solution using virtually any typeof screening assay. In this embodiment, the high degree of specificityof enzyme reactions on functional groups allows for the “tracking” ofspecific enzymatic reactions that make up the biocatalytically producedlibrary.

The invention also provides methods of discovering new phospholipasesusing the nucleic acids, polypeptides and antibodies of the invention.In one aspect, lambda phage libraries are screened for expression-baseddiscovery of phospholipases. Use of lambda phage libraries in screeningallows detection of toxic clones; improved access to substrate; reducedneed for engineering a host, by-passing the potential for any biasresulting from mass excision of the library; and, faster growth at lowclone densities. Screening of lambda phage libraries can be in liquidphase or in solid phase. Screening in liquid phase gives greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

In alternative embodiments, procedural steps are performed using roboticautomation; e.g., enabling the execution of many thousands ofbiocatalytic reactions and screening assays per day as well as ensuringa high level of accuracy and reproducibility (see discussion of arrays,below). As a result, a library of derivative compounds can be producedin a matter of weeks. For further teachings on modification ofmolecules, including small molecules, see PCT/US94/09174.

Phospholipase Signal Sequences

The invention provides phospholipase signal sequences (e.g., signalpeptides (SPs)), e.g., peptides comprising signal sequences and/orchimeric polypeptides, where the peptides or chimerics have a signalsequence as described herein. The invention provides nucleic acidsencoding these signal sequences (SPs, e.g., a peptide having a sequencecomprising/consisting of amino terminal residues of a polypeptide of theinvention). In one aspect, the invention provides a signal sequencecomprising a peptide comprising/consisting of a sequence as set forth inresidues 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26,1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32 or 1 to 33 of apolypeptide of the invention, e.g., a polypeptide comprising a sequenceas set forth in SEQ ID NO:6 and having one or more mutations as setforth in Tables 12 to 15, or an enzymatically active fragment thereof.Any of these peptides can be part of a chimeric protein, e.g., arecombinant protein. A signal sequence peptide can be matched withanother enzyme of the invention (e.g., a phospholipase of the inventionfrom which is was not derived), or, with another phospholipase, or withany polypeptide, as discussed further, below.

Exemplary signal sequences include residues 1 to 37 of SEQ ID NO:4 andresidues 1 to 23 of SEQ ID NO:6.

In some aspects phospholipases of the invention do not have signalsequences. In one aspect, the invention provides the phospholipases ofthe invention lacking all or part of a signal sequence. In one aspect,the invention provides a nucleic acid sequence encoding a signalsequence from one phospholipase operably linked to a nucleic acidsequence of a different phospholipase or, optionally, a signal sequencefrom a non-phospholipase protein may be desired.

Phospholipase Prepro Domains, Binding Domains and Catalytic Domains

In addition to signal sequences (e.g., signal peptides (SPs)), asdiscussed above, the invention provides prepro domains, binding domains(e.g., substrate binding domain) and catalytic domains (CDs). The SPdomains, binding domains, prepro domains and/or CDs of the invention canbe isolated, synthetic or recombinant peptides or can be part of afusion protein, e.g., as a heterologous domain in a chimeric protein.The invention provides nucleic acids encoding these catalytic domains(CDs) (e.g., “active sites”), prepro domains, binding domains and signalsequences (SPs, e.g., a peptide having a sequence comprising/consistingof amino terminal residues of a polypeptide of the invention).

The phospholipase signal sequences (SPs), binding domains, catalyticdomains (CDs) and/or prepro sequences of the invention can be isolatedpeptides, or, sequences joined to another phospholipase or anon-phospholipase polypeptide, e.g., as a fusion (chimeric) protein. Inone aspect, polypeptides comprising phospholipase signal sequences SPsand/or prepro of the invention comprise sequences heterologous tophospholipases of the invention (e.g., a fusion protein comprising an SPand/or prepro of the invention and sequences from another phospholipaseor a non-phospholipase protein). In one aspect, the invention providesphospholipases of the invention with heterologous CDs, SPs and/or preprosequences, e.g., sequences with a yeast signal sequence. A phospholipaseof the invention can comprise a heterologous CD, SP and/or prepro in avector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs, CDs, and/or prepro sequences of the invention areidentified following identification of novel phospholipase polypeptides.The pathways by which proteins are sorted and transported to theirproper cellular location are often referred to as protein targetingpathways. One of the most important elements in all of these targetingsystems is a short amino acid sequence at the amino terminus of a newlysynthesized polypeptide called the signal sequence. This signal sequencedirects a protein to its appropriate location in the cell and is removedduring transport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length from 13to 45 or more amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel hydrolase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

In some aspects, a phospholipase of the invention may not have SPsand/or prepro sequences, and/or catalytic domains (CDs). In one aspect,the invention provides phospholipases lacking all or part of an SP, a CDand/or a prepro domain. In one aspect, the invention provides a nucleicacid sequence encoding a signal sequence (SP), a CD and/or prepro fromone phospholipase operably linked to a nucleic acid sequence of adifferent phospholipase or, optionally, a signal sequence (SPs), a CDand/or prepro domain from a non-phospholipase protein may be desired.

The invention also provides isolated, synthetic or recombinantpolypeptides comprising signal sequences (SPs), prepro domain and/orcatalytic domains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa phospholipase) with an SP, prepro domain and/or CD. The sequence towhich the SP, prepro domain and/or CD are not naturally associated canbe on the SP's, prepro domain and/or CD's amino terminal end, carboxyterminal end, and/or on both ends of the SP and/or CD. In one aspect,the invention provides an isolated, synthetic or recombinant polypeptidecomprising (or consisting of) a polypeptide comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention withthe proviso that it is not associated with any sequence to which it isnaturally associated (e.g., phospholipase sequence). Similarly in oneaspect, the invention provides isolated, synthetic or recombinantnucleic acids encoding these polypeptides. Thus, in one aspect, theisolated, synthetic or recombinant nucleic acid of the inventioncomprises coding sequence for a signal sequence (SP), prepro domainand/or catalytic domain (CD) of the invention and a heterologoussequence (i.e., a sequence not naturally associated with the a signalsequence (SP), prepro domain and/or catalytic domain (CD) of theinvention). The heterologous sequence can be on the 3′ terminal end, 5′terminal end, and/or on both ends of the SP, prepro domain and/or CDcoding sequence.

The polypeptides of the invention include phospholipases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude phospholipases inactive for other reasons, e.g., before“activation” by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation, a de-glycosylation, a sulfation, adimerization event, and/or the like. Methods for identifying “prepro”domain sequences, CDs, binding domains and signal sequences are routineand well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136; yeast two-hybrid screenings for identifyingprotein-protein interactions, described e.g., by Miller (2004) MethodsMol. Biol. 261:247-62; Heyninck (2004) Methods Mol. Biol. 282:223-41,U.S. Pat. Nos. 6,617,122; 6,190,874. For example, to identify a preprosequence, the protein is purified from the extracellular space and theN-terminal protein sequence is determined and compared to theunprocessed form.

The polypeptides of the invention can be formulated as a proteinpreparation into any liquid, solid, semi-solid or gel form. For example,a protein preparation of the invention can comprise a formulationcomprising a non-aqueous liquid composition, a cast solid, a powder, alyophilized powder, a granular form, a particulate form, a compressedtablet, a pellet, a pill, a gel form, a hydrogel, a paste, an aerosol, aspray, a lotion or a slurry formulation.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains (CDs) or active sites, ofan enzyme of the invention. In one aspect, the invention providescatalytic domains or active sites as set forth below. In one aspect, theinvention provides a peptide or polypeptide comprising or consisting ofan active site domain as predicted through use of a database such asPfam (which is a large collection of multiple sequence alignments andhidden Markov models covering many common protein families, The Pfamprotein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin,L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall,and E. L. L. Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002) orequivalent.

The invention provides fusion of N-terminal or C-terminal subsequencesof enzymes of the invention (e.g., signal sequences, prepro sequences)with other polypeptides, active proteins or protein fragments. Theproduction of an enzyme of the invention (e.g., a phospholipase Cenzyme) may also be accomplished by expressing the enzyme as an inactivefusion protein that is later activated by a proteolytic cleavage event(using either an endogenous or exogenous protease activity, e.g.trypsin) that results in the separation of the fusion protein partnerand the mature enzyme, e.g., phospholipase C enzyme. In one aspect, thefusion protein of the invention is expressed from a hybrid nucleotideconstruct that encodes a single open reading frame containing thefollowing elements: the nucleotide sequence for the fusion protein, alinker sequence (defined as a nucleotide sequence that encodes aflexible amino acid sequence that joins two less flexible proteindomains), protease cleavage recognition site, and the mature enzyme(e.g., any enzyme of the invention, e.g., a phospholipase) sequence. Inalternative aspects, the fusion protein can comprise a pectate lyasesequence, a xylanase sequence, a phosphatidic acid phosphatase sequence,or another sequence, e.g., a sequence that has previously been shown tobe over-expressed in a host system of interest.

Any host system can be used (see discussion, above), for example, anybacteria, e.g., a gram positive bacteria, such as Bacillus, or a gramnegative bacteria, such as E. coli, or any yeast, e.g., Pichia pastoris.The arrangement of the nucleotide sequences in the chimeric nucleotideconstruction can be determined based on the protein expression levelsachieved with each fusion construct. Proceeding from the 5′ end of thenucleotide construct to the 3′ prime end of the construct, in oneaspect, the nucleotide sequences is assembled as follows: Signalsequence/fusion protein/linker sequence/protease cleavage recognitionsite/mature enzyme (e.g., any enzyme of the invention, e.g., aphospholipase) or Signal sequence/pro sequence/mature enzyme/linkersequence/fusion protein. The expression of enzyme (e.g., any enzyme ofthe invention, e.g., a phospholipase) as an inactive fusion protein mayimprove the overall expression of the enzyme's sequence, may reduce anypotential toxicity associated with the overproduction of active enzymeand/or may increase the shelf life of enzyme prior to use because enzymewould be inactive until the fusion protein e.g. pectate lyase isseparated from the enzyme, e.g., phospholipase protein.

In various aspects, the invention provides specific formulations for theactivation of phospholipase of the invention expressed as a fusionprotein. In one aspect, the activation of the phospholipase activityinitially expressed as an inactive fusion protein is accomplished usinga proteolytic activity or potentially a proteolytic activity incombination with an amino-terminal or carboxyl-terminal peptidase. Thisactivation event may be accomplished in a variety of ways and at varietyof points in the manufacturing/storage process prior to application inoil degumming. Exemplary processes of the invention include: Cleavage byan endogenous activity expressed by the manufacturing host uponsecretion of the fusion construct into the fermentation media; Cleavageby an endogenous protease activity that is activated or comes in contactwith intracellularly expressed fusion construct upon rupture of the hostcells; Passage of the crude or purified fusion construct over a columnof immobilized protease activity to accomplish cleavage and enzyme(e.g., phospholipase of the invention, e.g., a phospholipase C)activation prior to enzyme formulation; Treatment of the crude orpurified fusion construct with a soluble source of proteolytic activity;Activation of a phospholipase (e.g., a phospholipase of the invention,e.g., a phospholipase C) at the oil refinery using either a soluble orinsoluble source of proteolytic activity immediately prior to use in theprocess; and/or, Activation of the phospholipase (e.g., a phospholipaseof the invention, e.g., a phospholipase C) activity by continuouslycirculating the fusion construct formulation through a column ofimmobilized protease activity at reduced temperature (for example, anybetween about 4° C. and 20° C.). This activation event may beaccomplished prior to delivery to the site of use or it may occuron-site at the oil refinery.

Glycosylation

The peptides and polypeptides of the invention (e.g., hydrolases,antibodies) can also be glycosylated, for example, in one aspect,comprising at least one glycosylation site, e.g., an N-linked orO-linked glycosylation. In one aspect, the polypeptide can beglycosylated after being expressed in a P. pastoris or a S. pombe. Theglycosylation can be added post-translationally either chemically or bycellular biosynthetic mechanisms, wherein the later incorporates the useof known glycosylation motifs, which can be native to the sequence orcan be added as a peptide or added in the nucleic acid coding sequence.

Assays for Phospholipase Activity

The invention provides isolated, synthetic or recombinant polypeptides(e.g., enzymes, antibodies) having a phospholipase activity, or anycombination of phospholipase activities, and nucleic acids encodingthem. Any of the many phospholipase activity assays known in the art canbe used to determine if a polypeptide has a phospholipase activity andis within the scope of the invention. Routine protocols for determiningphospholipase A, B, D and C, patatin and lipid acyl hydrolaseactivities, or lipase activity, are well known in the art.

Exemplary activity assays include turbidity assays, methylumbelliferylphosphocholine (fluorescent) assays, Amplex red (fluorescent)phospholipase assays, thin layer chromatography assays (TLC), cytolyticassays and p-nitrophenylphosphorylcholine assays. Using these assayspolypeptides, peptides or antibodies can be quickly screened for aphospholipase activity.

The phospholipase activity can comprise a lipid acyl hydrolase (LAH)activity. See, e.g., Jimenez (2001) Lipids 36:1169-1174, describing anoctaethylene glycol monododecyl ether-based mixed micellar assay fordetermining the lipid acyl hydrolase activity of a patatin. Pinsirodom(2000) J. Agric. Food Chem. 48:155-160, describes an exemplary lipidacyl hydrolase (LAH) patatin activity.

Turbidity assays to determine phospholipase activity are described,e.g., in Kauffmann (2001) “Conversion of Bacillus thermocatenulatuslipase into an efficient phospholipase with increased activity towardslong-chain fatty acyl substrates by directed evolution and rationaldesign,” Protein Engineering 14:919-928; Ibrahim (1995) “Evidenceimplicating phospholipase as a virulence factor of Candida albicans,”Infect. Immun. 63:1993-1998.

Methylumbelliferyl (fluorescent) phosphocholine assays to determinephospholipase activity are described, e.g., in Goode (1997) “Evidencefor cell surface and internal phospholipase activity in ascidian eggs,”Develop. Growth Differ. 39:655-660; Diaz (1999) “Directfluorescence-based lipase activity assay,” BioTechniques 27:696-700.

Amplex Red (fluorescent) Phospholipase Assays to determine phospholipaseactivity are available as kits, e.g., the detection ofphosphatidylcholine-specific phospholipase using an Amplex Redphosphatidylcholine-specific phospholipase assay kit from MolecularProbes Inc. (Eugene, Oreg.), according to manufacturer's instructions.Fluorescence is measured in a fluorescence microplate reader usingexcitation at 560±10 nm and fluorescence detection at 590±10 nm. Theassay is sensitive at very low enzyme concentrations.

Thin layer chromatography assays (TLC) to determine phospholipaseactivity are described, e.g., in Reynolds (1991) Methods in Enzymol.197:3-13; Taguchi (1975) “Phospholipase from Clostridium novyi typeA.I.,” Biochim. Biophys. Acta 409:75-85. Thin layer chromatography (TLC)is a widely used technique for detection of phospholipase activity.Various modifications of this method have been used to extract thephospholipids from the aqueous assay mixtures. In some PLC assays thehydrolysis is stopped by addition of chloroform/methanol (2:1) to thereaction mixture. The unreacted starting material and the diacylglycerolare extracted into the organic phase and may be fractionated by TLC,while the head group product remains in the aqueous phase. For moreprecise measurement of the phospholipid digestion, radiolabeledsubstrates can be used (see, e.g., Reynolds (1991) Methods in Enzymol.197:3-13). The ratios of products and reactants can be used to calculatethe actual number of moles of substrate hydrolyzed per unit time. If allthe components are extracted equally, any losses in the extraction willaffect all components equally. Separation of phospholipid digestionproducts can be achieved by silica gel TLC withchloroform/methanol/water (65:25:4) used as a solvent system (see, e.g.,Taguchi (1975) Biochim Biophys. Acta 409:75-85).

p-Nitrophenylphosphorylcholine assays to determine phospholipaseactivity are described, e.g., in Korbsrisate (1999) J. Clin. Microbiol.37:3742-3745; Berka (1981) Infect. Immun. 34:1071-1074. This assay isbased on enzymatic hydrolysis of the substrate analogp-nitrophenylphosphorylcholine to liberate a yellow chromogenic compoundp-nitrophenol, detectable at 405 nm. This substrate is convenient forhigh-throughput screening.

A cytolytic assay can detect phospholipases with cytolytic activitybased on lysis of erythrocytes. Toxic phospholipases can interact witheukaryotic cell membranes and hydrolyze phosphatidylcholine andsphingomyelin, leading to cell lysis. See, e.g., Titball (1993)Microbiol. Rev. 57:347-366.

Hybrid (Chimeric) Phospholipases and Peptide Libraries

In one aspect, the invention provides hybrid phospholipases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asphospholipase substrates, receptors, enzymes. The peptide libraries ofthe invention can be used to identify formal binding partners oftargets, such as ligands, e.g., cytokines, hormones and the like. In oneaspect, the invention provides chimeric proteins comprising a signalsequence (SP) and/or catalytic domain (CD) of the invention and aheterologous sequence (see above).

The invention also provides methods for generating “improved” and hybridphospholipases using the nucleic acids and polypeptides of theinvention. For example, the invention provides methods for generatingenzymes that have activity, e.g., phospholipase activity (such as, e.g.,phospholipase A, B, C or D activity, patatin esterase activity, cleavageof a glycerolphosphate ester linkage, cleavage of an ester linkage in aphospholipid in a vegetable oil) at extreme alkaline pHs and/or acidicpHs, high and low temperatures, osmotic conditions and the like. Theinvention provides methods for generating hybrid enzymes (e.g., hybridphospholipases).

In one aspect, the methods of the invention produce new hybridpolypeptides by utilizing cellular processes that integrate the sequenceof a first polynucleotide such that resulting hybrid polynucleotidesencode polypeptides demonstrating activities derived from the firstbiologically active polypeptides. For example, the first polynucleotidescan be an exemplary nucleic acid sequence encoding an exemplaryphospholipase of the invention. The first nucleic acid can encode anenzyme from one organism that functions effectively under a particularenvironmental condition, e.g. high salinity. It can be “integrated” withan enzyme encoded by a second polynucleotide from a different organismthat functions effectively under a different environmental condition,such as extremely high temperatures. For example, when the two nucleicacids can produce a hybrid molecule by e.g., recombination and/orreductive reassortment. A hybrid polynucleotide containing sequencesfrom the first and second original polynucleotides may encode an enzymethat exhibits characteristics of both enzymes encoded by the originalpolynucleotides. Thus, the enzyme encoded by the hybrid polynucleotidemay function effectively under environmental conditions shared by eachof the enzymes encoded by the first and second polynucleotides, e.g.,high salinity and extreme temperatures.

Alternatively, a hybrid polypeptide resulting from this method of theinvention may exhibit specialized enzyme activity not displayed in theoriginal enzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding phospholipase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized activities obtained from each of the originalenzymes, i.e. the type of bond on which the phospholipase acts and thetemperature at which the phospholipase functions. Thus, for example, thephospholipase may be screened to ascertain those chemicalfunctionalities which distinguish the hybrid phospholipase from theoriginal phospholipases, such as: (a) amide (peptide bonds), i.e.,phospholipases; (b) ester bonds, i.e., phospholipases and lipases; (c)acetals, i.e., glycosidases and, for example, the temperature, pH orsalt concentration at which the hybrid polypeptide functions.

Sources of the polynucleotides to be “integrated” with nucleic acids ofthe invention may be isolated from individual organisms (“isolates”),collections of organisms that have been grown in defined media(“enrichment cultures”), or, uncultivated organisms (“environmentalsamples”). The use of a culture-independent approach to derivepolynucleotides encoding novel bioactivities from environmental samplesis most preferable since it allows one to access untapped resources ofbiodiversity. “Environmental libraries” are generated from environmentalsamples and represent the collective genomes of naturally occurringorganisms archived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample that may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions that promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which hybrid polynucleotides may be preparedinclude prokaryotic microorganisms, such as Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. Polynucleotides may be isolated from environmentalsamples. Nucleic acid may be recovered without culturing of an organismor recovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles. Inone aspect, polynucleotides encoding phospholipase enzymes isolated fromextremophilic microorganisms are used to make hybrid enzymes. Suchenzymes may function at temperatures above 100° C. in, e.g., terrestrialhot springs and deep sea thermal vents, at temperatures below 0° C. in,e.g., arctic waters, in the saturated salt environment of, e.g., theDead Sea, at pH values around 0 in, e.g., coal deposits and geothermalsulfur-rich springs, or at pH values greater than 11 in, e.g., sewagesludge. For example, phospholipases cloned and expressed fromextremophilic organisms can show high activity throughout a wide rangeof temperatures and pHs.

Polynucleotides selected and isolated as described herein, including atleast one nucleic acid of the invention, are introduced into a suitablehost cell. A suitable host cell is any cell that is capable of promotingrecombination and/or reductive reassortment. The selectedpolynucleotides can be in a vector that includes appropriate controlsequences. The host cell can be a higher eukaryotic cell, such as amammalian cell, or a lower eukaryotic cell, such as a yeast cell, orpreferably, the host cell can be a prokaryotic cell, such as a bacterialcell. Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-Dextran mediated transfection,or electroporation (Davis et al., 1986).

Exemplary appropriate hosts may be any of the host cells familiar tothose skilled in the art, including prokaryotic cells, eukaryotic cells,such as bacterial cells, fungal cells, yeast cells, mammalian cells,insect cells, or plant cells. Exemplary bacterial cells include anyspecies within the genera Escherichia, Bacillus, Streptomyces,Salmonella, Pseudomonas and Staphylococcus, including, e.g., Escherichiacoli, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonellatyphimurium, Pseudomonas fluorescens. Exemplary fungal cells include anyspecies of Aspergillus. Exemplary yeast cells include any species ofPichia, Saccharomyces, Schizosaccharomyces, or Schwanniomyces, includingPichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.Exemplary insect cells include any species of Spodoptera or Drosophila,including Drosophila S2 and Spodoptera Sf9. Exemplary animal cellsinclude CHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. The selection of an appropriate host forrecombination and/or reductive reassortment or just for expression ofrecombinant protein is deemed to be within the scope of those skilled inthe art from the teachings herein. Mammalian cell culture systems thatcan be employed for recombination and/or reductive reassortment or justfor expression of recombinant protein include, e.g., the COS-7 lines ofmonkey kidney fibroblasts, described in “SV40-transformed simian cellssupport the replication of early SV40 mutants” (Gluzman, 1981), theC127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectorscan comprise an origin of replication, a suitable promoter and enhancer,and necessary ribosome binding sites, polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

Host cells containing the polynucleotides of interest (for recombinationand/or reductive reassortment or just for expression of recombinantprotein) can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan. Theclones which are identified as having the specified enzyme activity maythen be sequenced to identify the polynucleotide sequence encoding anenzyme having the enhanced activity.

In another aspect, the nucleic acids and methods of the presentinvention can be used to generate novel polynucleotides for biochemicalpathways, e.g., pathways from one or more operons or gene clusters orportions thereof. For example, bacteria and many eukaryotes have acoordinated mechanism for regulating genes whose products are involvedin related processes. The genes are clustered, in structures referred toas “gene clusters,” on a single chromosome and are transcribed togetherunder the control of a single regulatory sequence, including a singlepromoter which initiates transcription of the entire cluster. Thus, agene cluster is a group of adjacent genes that are either identical orrelated, usually as to their function.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples.“Fosmids,” cosmids or bacterial artificial chromosome (BAC) vectors canbe used as cloning vectors. These are derived from E. coli f-factorwhich is able to stably integrate large segments of genomic DNA. Whenintegrated with DNA from a mixed uncultured environmental sample, thismakes it possible to achieve large genomic fragments in the form of astable “environmental DNA library.” Cosmid vectors were originallydesigned to clone and propagate large segments of genomic DNA. Cloninginto cosmid vectors is described in detail in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989). Once ligated into an appropriate vector, two or morevectors containing different polyketide synthase gene clusters can beintroduced into a suitable host cell. Regions of partial sequencehomology shared by the gene clusters will promote processes which resultin sequence reorganization resulting in a hybrid gene cluster. The novelhybrid gene cluster can then be screened for enhanced activities notfound in the original gene clusters.

Thus, in one aspect, the invention relates to a method for producing abiologically active hybrid polypeptide using a nucleic acid of theinvention and screening the polypeptide for an activity (e.g., enhancedactivity) by:

-   -   (1) introducing at least a first polynucleotide (e.g., a nucleic        acid of the invention) in operable linkage and a second        polynucleotide in operable linkage, said at least first        polynucleotide and second polynucleotide sharing at least one        region of partial sequence homology, into a suitable host cell;    -   (2) growing the host cell under conditions which promote        sequence reorganization resulting in a hybrid polynucleotide in        operable linkage;    -   (3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   (4) screening the hybrid polypeptide under conditions which        promote identification of the desired biological activity (e.g.,        enhanced phospholipase activity); and    -   (5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

In vivo reassortment can be focused on “inter-molecular” processescollectively referred to as “recombination.” In bacteria it is generallyviewed as a “RecA-dependent” phenomenon. The invention can rely onrecombination processes of a host cell to recombine and re-assortsequences, or the cells' ability to mediate reductive processes todecrease the complexity of quasi-repeated sequences in the cell bydeletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process. Thus, in one aspect of theinvention, using the nucleic acids of the invention novelpolynucleotides are generated by the process of reductive reassortment.The method involves the generation of constructs containing consecutivesequences (original encoding sequences), their insertion into anappropriate vector, and their subsequent introduction into anappropriate host cell. The reassortment of the individual molecularidentities occurs by combinatorial processes between the consecutivesequences in the construct possessing regions of homology, or betweenquasi-repeated units. The reassortment process recombines and/or reducesthe complexity and extent of the repeated sequences, and results in theproduction of novel molecular species.

Various treatments may be applied to enhance the rate of reassortment.These could include treatment with ultra-violet light, or DNA damagingchemicals, and/or the use of host cell lines displaying enhanced levelsof “genetic instability”. Thus the reassortment process may involvehomologous recombination or the natural property of quasi-repeatedsequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. “Quasi-repeats” are repeats that are not restricted totheir original unit structure. Quasi-repeated units can be presented asan array of sequences in a construct; consecutive units of similarsequences. Once ligated, the junctions between the consecutive sequencesbecome essentially invisible and the quasi-repetitive nature of theresulting construct is now continuous at the molecular level. Thedeletion process the cell performs to reduce the complexity of theresulting construct operates between the quasi-repeated sequences. Thequasi-repeated units provide a practically limitless repertoire oftemplates upon which slippage events can occur. The constructscontaining the quasi-repeats thus effectively provide sufficientmolecular elasticity that deletion (and potentially insertion) eventscan occur virtually anywhere within the quasi-repetitive units. When thequasi-repeated sequences are all ligated in the same orientation, forinstance head to tail or vice versa, the cell cannot distinguishindividual units. Consequently, the reductive process can occurthroughout the sequences. In contrast, when for example, the units arepresented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, in one aspect of theinvention, the sequences to be reassorted are in the same orientation.Random orientation of quasi-repeated sequences will result in the lossof reassortment efficiency, while consistent orientation of thesequences will offer the highest efficiency. However, while having fewerof the contiguous sequences in the same orientation decreases theefficiency, it may still provide sufficient elasticity for the effectiverecovery of novel molecules. Constructs can be made with thequasi-repeated sequences in the same orientation to allow higherefficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following: a) Primers that include apoly-A head and poly-T tail which when made single-stranded wouldprovide orientation can be utilized. This is accomplished by having thefirst few bases of the primers made from RNA and hence easily removedRNase H. b) Primers that include unique restriction cleavage sites canbe utilized. Multiple sites, a battery of unique sequences, and repeatedsynthesis and ligation steps would be required. c) The inner few basesof the primer could be thiolated and an exonuclease used to produceproperly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (R1). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by: 1) The use of vectors only stablymaintained when the construct is reduced in complexity. 2) The physicalrecovery of shortened vectors by physical procedures. In this case, thecloning vector would be recovered using standard plasmid isolationprocedures and size fractionated on either an agarose gel, or columnwith a low molecular weight cut off utilizing standard procedures. 3)The recovery of vectors containing interrupted genes which can beselected when insert size decreases. 4) The use of direct selectiontechniques with an expression vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, this process is not limitedto such nearly identical repeats.

The following is an exemplary method of the invention. Encoding nucleicacid sequences (quasi-repeats) are derived from three (3) species,including a nucleic acid of the invention. Each sequence encodes aprotein with a distinct set of properties, including an enzyme of theinvention. Each of the sequences differs by a single or a few base pairsat a unique position in the sequence. The quasi-repeated sequences areseparately or collectively amplified and ligated into random assembliessuch that all possible permutations and combinations are available inthe population of ligated molecules. The number of quasi-repeat unitscan be controlled by the assembly conditions. The average number ofquasi-repeated units in a construct is defined as the repetitive index(R1). Once formed, the constructs may, or may not be size fractionatedon an agarose gel according to published protocols, inserted into acloning vector, and transfected into an appropriate host cell. The cellsare then propagated and “reductive reassortment” is effected. The rateof the reductive reassortment process may be stimulated by theintroduction of DNA damage if desired. Whether the reduction in RI ismediated by deletion formation between repeated sequences by an“intra-molecular” mechanism, or mediated by recombination-like eventsthrough “inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations. In oneaspect, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure. The polypeptides, e.g.,phospholipases, that are identified from such libraries can be used forvarious purposes, e.g., the industrial processes described herein and/orcan be subjected to one or more additional cycles of shuffling and/orselection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)—CC-1065, or a synthetic analog such as (+)—CC-1065-(N3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred means for slowing or haltingPCR amplification consist of UV light (+)—CC-1065 and(+)—CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forphospholipase activity, to screen compounds as potential modulators ofactivity (e.g., potentiation or inhibition of enzyme activity), forantibodies that bind to a polypeptide of the invention, for nucleicacids that hybridize to a nucleic acid of the invention, and the like.

Immobilized Enzyme Solid Supports

The phospholipase enzymes, fragments thereof and nucleic acids thatencode the enzymes and fragments can be affixed to a solid support. Thisis often economical and efficient in the use of the phospholipases inindustrial processes. For example, a consortium or cocktail ofphospholipase enzymes (or active fragments thereof), which are used in aspecific chemical reaction, can be attached to a solid support anddunked into a process vat. The enzymatic reaction can occur. Then, thesolid support can be taken out of the vat, along with the enzymesaffixed thereto, for repeated use. In one embodiment of the invention,an isolated nucleic acid of the invention is affixed to a solid support.In another embodiment of the invention, the solid support is selectedfrom the group of a gel, a resin, a polymer, a ceramic, a glass, amicroelectrode and any combination thereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include Sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,AI₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support that canbe used is a microelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support.

Other exemplary solid supports used to practice the invention comprisediatomaceous earth products and silicates. Some examples include CELITE®KENITE®, DIACTIV®, PRIMISIL®, DIAFIL® diatomites and MICRO-CEL®,CALFLO®, SILASORB™, and CELKATE® synthetic calcium and magnesiumsilicates. Another example of a solid support is a cell, such as a redblood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the artfor immobilizing enzymes or fragments thereof, or nucleic acids, onto asolid support. Some examples of such methods include, e.g.,electrostatic droplet generation, electrochemical means, via adsorption,via covalent binding, via cross-linking, via a chemical reaction orprocess, via encapsulation, via entrapment, via calcium alginate, or viapoly(2-hydroxyethyl methacrylate). Like methods are described in Methodsin Enzymology, Immobilized Enzymes and Cells, Part C. 1987. AcademicPress. Edited by S. P. Colowick and N, O. Kaplan. Volume 136; andImmobilization of Enzymes and Cells. 1997. Humana Press. Edited by G. F.Bickerstaff. Series: Methods in Biotechnology, Edited by J. M. Walker.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into afirst component into at least a portion of a capillary of a capillaryarray. Each capillary of the capillary array can comprise at least onewall defining a lumen for retaining the first component. An air bubblecan be introduced into the capillary behind the first component. Asecond component can be introduced into the capillary, wherein thesecond component is separated from the first component by the airbubble. A sample of interest can be introduced as a first liquid labeledwith a detectable particle into a capillary of a capillary array,wherein each capillary of the capillary array comprises at least onewall defining a lumen for retaining the first liquid and the detectableparticle, and wherein the at least one wall is coated with a bindingmaterial for binding the detectable particle to the at least one wall.The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.

The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a microtiter plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “BioChips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a phospholipase gene. One or more, or, all the transcripts of a cellcan be measured by hybridization of a sample comprising transcripts ofthe cell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. “Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins.

In alternative embodiment, the invention provides “arrays” or“microarrays” or “biochips” or “chips” comprising a plurality of targetelements, wherein each target element can comprise a defined amount ofone or more polypeptides (including antibodies) or nucleic acidsimmobilized onto a defined area of a substrate surface, and at least onenucleic acid and/or polypeptide is a nucleic acid and/or polypeptide ofthis invention.

The present invention can be practiced with, or can comprise, any known“array,” also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a phospholipase of the invention. Theseantibodies can be used to isolate, identify or quantify thephospholipases of the invention or related polypeptides. Theseantibodies can be used to inhibit the activity of an enzyme of theinvention. These antibodies can be used to isolated polypeptides relatedto those of the invention, e.g., related phospholipase enzymes.

An “antibody” of this invention can include a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope, see, e.g.Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

The antibodies can be used in immunoprecipitation, staining (e.g.,FACS), immunoaffinity columns, and the like. If desired, nucleic acidsequences encoding for specific antigens can be generated byimmunization followed by isolation of polypeptide or nucleic acid,amplification or cloning and immobilization of polypeptide onto an arrayof the invention.

Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides can be used to generate antibodies which bindspecifically to the polypeptides of the invention. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to an animal, forexample, a nonhuman. The antibody so obtained will then bind thepolypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies which maybind to the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides from other organisms andsamples. In such techniques, polypeptides from the organism arecontacted with the antibody and those polypeptides which specificallybind the antibody are detected. Any of the procedures described abovemay be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, polypeptides (e.g., a kithaving at least one phospholipase of the invention) and/or antibodies(e.g., a kit having at least one antibody of the invention. The kits cancontain enzymes for the processing (the making of) biofuels, detergents,or for treating or processing foods, feeds, biomass, food or feedadditives or nutritional supplements, and the like. The kits also cancontain instructional material teaching the methodologies and industrialuses of the invention, as described herein.

Industrial and Medical Uses of the Enzymes of the Invention

The invention provides many industrial uses and medical applicationsusing polypeptides of the invention, e.g., a phospholipase and otherenzymes of the invention, e.g., phospholipases A, B, C and D, patatins,including converting a non-hydratable phospholipid to a hydratable form,making biofuels and processing biomass, oil degumming, processing ofoils from plants, fish, algae and the like, to name just a fewapplications. In any of these alternative industrial uses and medicalapplications, an enzymes can be added in a specific order, e.g.,phospholipases with differing specificities are added in a specificorder, for example, an enzyme with PC- and PE-hydrolyzing activity isadded first (or two enzymes are added, one with PC-hydrolyzing activityand the other with PE-hydrolyzing activity), then an enzyme withPI-hydrolyzing activity (e.g., PLC or PI-PLC activity) is added, or anycombination thereof.

Any or all of the methods of the invention can be used on a “processscale”, e.g., an oil processes or refining on a scale from about 15,000;25,000; 50,000; 75,000; or 100,000 lbs of refined oil/day up to about 1,2, 3, 4, 5 or 6 or more million lbs refined oil/day.

Methods of using phospholipase enzymes in industrial applications arewell known in the art. For example, the phospholipases and methods ofthe invention can be used for the processing of fats and oils asdescribed, e.g., in JP Patent Application Publication H6-306386,describing converting phospholipids present in the oils and fats intowater-soluble substances containing phosphoric acid groups.

Phospholipases of the invention can be used to process plant oils andphospholipids such as those derived from or isolated from rice bran,soy, canola, palm, cottonseed, corn, palm kernel, coconut, peanut,sesame, sunflower. Phospholipases of the invention can be used toprocess essential oils, e.g., those from fruit seed oils, e.g.,grapeseed, apricot, borage, etc. Phospholipases of the invention can beused to process oils and phospholipids in different forms, includingcrude forms, degummed, gums, wash water, clay, silica, soapstock, andthe like. The phospholipids of the invention can be used to process highphosphorus oils, fish oils, animal oils, plant oils, algae oils and thelike. In any aspect of the invention, any time a phospholipase C can beused, an alternative comprises use of a phospholipase D of the inventionand a phosphatase (e.g., using a PLD/phosphatase combination to improveyield in a high phosphorus oil, such as a soy bean oil).

Phospholipases of the invention can be used to process and make edibleoils, biodiesel oils, liposomes for pharmaceuticals and cosmetics,structured phospholipids and structured lipids. Phospholipases of theinvention can be used in oil extraction. Phospholipases of the inventioncan be used to process and make various soaps.

In another embodiment, provided herein is a method for obtaining aphospholipid from an edible oil. In certain embodiment, thephospholipids obtained by the methods provided herein include a varietyof phospholipids, including, but not limited to phosphatidylcholine(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylinositol (PI), phosphatidic acid (PA),lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE),lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI),lysophosphatidic acid (LPA), choline (C), ethanolamine (E), serine (S),and inositol (I).

Processing Edible Oils: Generation of 1,3-diacylglycerol (1,3 DAG)

The invention provides processes using enzyme(s) of the invention tomake 1,3-diacylglycerol (1,3 DAG). In one aspect, a phospholipase C orphospholipase D plus a phosphatase generates 1,2-diacylglycerol; thisimproves oil yield during edible oil refining. When used in a processthat includes a caustic neutralization step, for example as a causticrefining aid, as much as 70% of the 1,2-diacylglyceride (1,2-DAG)undergoes acyl migration and is converted to 1,3-DAG. 1,3-DAG possessesincreased health benefits and therefore the use of PLC as a causticrefining aid produces an oil with increased nutritional value.

The invention provides processes using enzyme(s) of the invention tomake and process edible oils, including generation of edible oils withincreased amounts of 1,3-DAG. Diacylglycerols are naturally occurringcompounds found in many edible oils. In one aspect of a method of theinvention, e.g., the oil degumming process, a base (caustic) causes theisomerization of 1,2-DAG, produced by PLC, into 1,3-DAG which provides anutritional health benefit over 1,2-DAG, e.g., the 1,3-DAG is burned asenergy instead of being stored as fat (as is 1,2-DAG). By adding the PLCat the front end of caustic refining process (and the acid and causticsubsequently), the methods of the invention generate an elevated levelof 1,3-DAG (decreasing 1,2-DAG). Nutritionally, 1,3-DAG is better foryou than 1,2-DAG. In alternative aspects, the invention comprises an oildegumming process using a PLC of the invention, whereby the finaldegummed oil product contains not less than 0.5%, 1.0%, 2.0% or 3.0% ormore 1,3-DAG.

Thus, the invention provides a process for making (throughinteresterification) a refined oil (e.g., a diacylglycerol oil),including edible oils, containing increased levels of 1,3-diacylglycerol(1,3-DAG), where a phospholipase, such as an enzyme of the invention, is“front-loaded” or added before addition of acid or caustic. Thegeneration by enzymatic hydrolysis of a DAG from a triglyceridegenerates by interesterification 1,3 DAG from 1,2 DAG. The 1,3DAG-comprising edible oil shows different metabolic effects compared toconventional edible oils. Differences in metabolic pathways between 1,3DAG and either 1,2 DAG or triglycerides allow a greater portion of fattyacids from 1,3 diacylglycerol to be burned as energy rather than beingstored as fat. Clinical studies have shown that regular consumption ofDAG oil as part of a sensible diet can help individuals to manage theirbody weight and body fat. In addition, metabolism of 1,3 DAG reducescirculating postmeal triglycerides in the bloodstream. Since obesity andelevated blood lipids are associated as risk factors for chronicdiseases including cardiovascular disease and Type II diabetes, theselifestyle-related health conditions may be impacted in a beneficialmanner with regular consumption of DAG oils.

Consumption of DAG-comprising oil can take place through a variety ofmeans. Thus, in one aspect, the invention provides a process using anenzyme of the invention for making a food, e.g., a baked good, havingincreased levels of 1,3-DAG diacylglycerol and baked goods comprisingdiacylglycerol oils. In one aspect, the baked goods are cookies, cakesand similar baked goods.

In alternative embodiments, combination of enzymes that can be used inthe methods of the invention, including the processing of edible oils,include (where one, several or all of the enzymes in the combinationcomprise an enzyme of the instant invention):

-   -   PLC+PI-PLC+PLA (PLA added after completion of PLC reactions);    -   PLD+phosphatase+PI-PLC followed by PLA; or,    -   PLC or (PLC+PI-PLC)+PLA specific for phosphatidic acid (all        enzymes added together or sequentially).

Oil Degumming and Vegetable Oil Processing

The enzymes of the invention (e.g., polypeptides of the invention havinglipase, phospholipase, esterase and/or glycosidase or equivalentactivity) can be used in various vegetable oil processing steps, such asin vegetable oil extraction, particularly, in the removal of“phospholipid gums” in a process called “oil degumming”.

These processes of the invention can be used on a “process scale”, e.g.,on a scale from about 15,000; 25,000; 50,000; 75,000; or 100,000 lbs ofrefined oil/day up to about 1, 2, 3, 4, 5 or 6 or more million lbsrefined oil/day.

In one aspect, the invention provides oil degumming processes comprisinguse of a phospholipase of the invention, e.g., a PLC, e.g. a PI-PLC ofthe invention. In one aspect, the process further comprises addition ofanother phospholipase (which can also be a phospholipase of theinvention), e.g., another PLC, a PLA, a PLB, a PLB or a patatin of theinvention, or an enzyme (which can also be an enzyme of the invention)having a lysophospholipase-transacylase (LPTA) activity orlysophospholipase (LPL) activity and lysophospholipase-transacylase(LPTA), or a combination thereof, and/or a patatin-like phospholipase(which can also be an enzyme of the invention). In one aspect, allenzymes are added together, or, alternatively, the enzymes are added ina specific order, e.g., PLC addition is followed by PLA and/or patatinaddition; or, an enzyme or enzymes of the invention having PC and PEactivity added first, then PI PLC added second.

In one aspect, this process provides a yield improvement as a result ofthe phospholipase (e.g., PLC of the invention) treatment. In one aspect,this process provides an additional decrease of the phosphorus contentof the oil as a result of the phospholipase (e.g., PLA of the invention)treatment.

In one aspect, the invention provides processes comprising use of aphospholipase of the invention, e.g., a PLC or a PI-PLC of theinvention, to reduce gum mass and increase neutral oil (triglyceride)gain through reduced oil entrapment. In one aspect, the inventionprovides processes comprising use of a phospholipase of the invention,e.g., a PLC of the invention, e.g., a PI-PLC of the invention, forincreasing neutral oils and diacylglycerol (DAG) production tocontribute to the oil phase. In alternative aspects, processes of theinvention (e.g., degumming processes) may comprise one or more otherenzymes such as a protease, an amylase, a lipase, a cutinase, anotherphospholipase (including, e.g., an enzyme of the invention), acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase, or polypeptides with equivalent activity, or a combinationthereof.

The phospholipases of the invention can be used in various vegetable oilprocessing steps, such as in vegetable oil extraction, particularly, inthe removal of “phospholipid gums” in a process called “oil degumming,”as described above. The invention provides methods for processingvegetable oils from various sources, such as rice bran, soybeans,rapeseed, peanuts and other nuts, sesame, sunflower, palm and corn. Themethods can used in conjunction with processes based on extraction withas hexane, with subsequent refining of the crude extracts to edibleoils, including use of the methods and enzymes of the invention. Thefirst step in the refining sequence is the so-called “degumming”process, which serves to separate phosphatides by the addition of water.The material precipitated by degumming is separated and furtherprocessed to mixtures of lecithins. The commercial lecithins, such assoybean lecithin and sunflower lecithin, are semi-solid or very viscousmaterials. They consist of a mixture of polar lipids, mainlyphospholipids, and oil, mainly triglycerides.

The phospholipases of the invention can be used in any “degumming”procedure, including water degumming, ALCON oil degumming (e.g., forsoybeans), safinco degumming, “super degumming,” UF degumming, TOPdegumming, uni-degumming, dry degumming and ENZYMAX™ degumming. See,e.g., U.S. Pat. Nos. 6,355,693; 6,162,623; 6,103,505; 6,001,640;5,558,781; 5,264,367. Various “degumming” procedures incorporated by themethods of the invention are described in Bockisch, M. (1998) In Fatsand Oils Handbook, The extraction of Vegetable Oils (Chapter 5),345-445, AOCS Press, Champaign, Ill. The phospholipases of the inventioncan be used in the industrial application of enzymatic degumming oftriglyceride oils as described, e.g., in EP 513 709.

In one aspect, phospholipases of the invention are used to treatvegetable oils, e.g., crude oils, such as rice bran, soy, canola, flowerand the like. In one aspect, this improves the efficiency of thedegumming process. In one aspect, the invention provides methods forenzymatic degumming under conditions of low water, e.g., in the range ofbetween about 0.1% to 20% water, or, 0.5% to 10% water. In one aspect,this results in the improved separation of a heavy phase from the oilphase during centrifugation. The improved separation of these phases canresult in more efficient removal of phospholipids from the oil,including both hydratable and nonhydratable oils. In one aspect, thiscan produce a gum fraction that contains less entrained neutral oil(triglycerides), thereby improving the overall yield of oil during thedegumming process.

In one aspect, phospholipases of the invention, e.g., a polypeptidehaving PLC activity, e.g., a PI-PLC activity, are used to treat oils(e.g., vegetable oils, including crude oils, such as rice bran, soy,canola, flower and the like), e.g., in degumming processes, to reducegum mass and increase neutral oil gain through reduced oil entrapment.In one aspect, phospholipases of the invention e.g., a polypeptidehaving PLC activity, are used for diacylglycerol (DAG) production and tocontribute to the oil phase.

The phospholipases of the invention can be used in the industrialapplication of enzymatic degumming as described, e.g., in CA 1102795,which describes a method of isolating polar lipids from cereal lipids bythe addition of at least 50% by weight of water. This method is amodified degumming in the sense that it utilizes the principle of addingwater to a crude oil mixture.

In one aspect, the invention provides enzymatic processes comprising useof phospholipases of the invention (e.g., a PLC, e.g., a PI-PLC)comprising hydrolysis of hydrated phospholipids in oil at a temperatureof about 20° C. to 40° C., at an alkaline pH, e.g., a pH of about pH 8to pH 10, using a reaction time of about 3 to 10 minutes. This canresult in less than 10 ppm final oil phosphorus levels. The inventionalso provides enzymatic processes comprising use of phospholipases ofthe invention (e.g., a PI-PLC) comprising hydrolysis of hydratable andnon-hydratable phospholipids in oil at a temperature of about 50° C. to60° C., at a pH slightly below neutral, e.g., of about pH 5 to pH 6.5,using a reaction time of about 30 to 60 minutes. This can result in lessthan 10 ppm final oil phosphorus levels.

In one aspect, the invention provides enzymatic processes that utilize aphospholipase C enzyme to hydrolyze a glyceryl phosphoester bond andthereby enable the return of the diacylglyceride portion ofphospholipids back to the oil, e.g., a vegetable, fish or algae oil (a“phospholipase C (PLC) caustic refining aid”); and, reduce thephospholipid content in a degumming step to levels low enough for highphosphorus oils to be physically refined (a “phospholipase C (PLC)degumming aid”). The two approaches can generate different values andhave different target applications.

In various exemplary processes of the invention, a number of distinctsteps compose the degumming process preceding the core bleaching anddeodorization refining processes. These steps include heating, mixing,holding, separating and drying. Following the heating step, water andoften acid are added and mixed to allow the insoluble phospholipid “gum”to agglomerate into particles which may be separated. While waterseparates many of the phosphatides in degumming, portions of thephospholipids are non-hydratable phosphatides (NHPs) present as calciumor magnesium salts. Degumming processes address these NHPs by theaddition of acid. Following the hydration of phospholipids, the oil ismixed, held and separated by centrifugation. Finally, the oil is driedand stored, shipped or refined, as illustrated, e.g., in FIG. 1. Theresulting gums are either processed further for lecithin products oradded back into the meal.

In one embodiment, provided herein is a method for hydration of nonhydratable phospholipids within a lipid matrix by enabling them tomigrate to an oil-water interface. The non hydratable phospholipids arethen reacted and/or removed from the lipids. In one embodiment, themethod comprises a) mixing an aqueous acid with an edible oil to obtainan acidic mixture having pH of less than about 4; and b) mixing a basewith the acidic mixture to obtain a reacted mixture having pH of about6-9, wherein the mixing in steps a) and/or b) creates an emulsion thatcomprises an aqueous phase in average droplet size between about 15 μmto about 45 μm in size. In certain embodiments, mixing in steps a)and/or b) creates an emulsion that comprises at least about 60% of anaqueous phase by volume in droplet size between about 15 μm to about 45μm in size, wherein percentage of the aqueous phase is based on thetotal volume of the aqueous phase. In certain embodiment, the methodsprovided herein allow the non hydratable phospholipids within a lipidmatrix to migrate to an oil-water interface.

In certain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises an aqueous phase in average droplet size betweenabout 15-40 μm, 15-35 μm, 17-40 μm, 20-40 μm, 20-30 μm, 25-30 μm, 25-40μm, or 25-35 μm. In certain embodiments, the mixing in step a) createsan emulsion that comprises an aqueous phase in average droplet sizebetween about 15-40 μm, 15-35 μm, 17-40 μm, 20-40 μm, 20-30 μm, 25-30μm, 25-40 μm, or 25-35 μm. In certain embodiments, the mixing in step b)creates an emulsion that comprises an aqueous phase in average dropletsize between about 15-40 μm, 15-35 μm, 17-40 μm, 20-40 μm, 20-30 μm,25-30 μm, 25-40 μm, or 25-35 μm. In certain embodiments, the averagedroplet size is about 15 μm, 17 μm, 19 μm, 20 μm, 22 μm, 25 μm, 27 μm,30 μm, 35 μm, or 40 μm. In certain embodiments, the average droplet sizeis about 20 μm.

In certain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 60% of the aqueous phase byvolume in droplet size between about 20 μm to about 40 μm in size,wherein percentage of the aqueous phase is based on the total volume ofthe aqueous phase. In certain embodiments, the mixing steps creates anemulsion that comprises about 60-95%, 60-90%, 60-80%, 70-95%, 80-95% ofthe aqueous phase by volume in droplet size between about 20-40 μm,20-35 μm, 25-40 μm, 30-40 μm, 35-40 μm, or 25-45 μm, wherein percentageof the aqueous phase is based on the total volume of the aqueous phase.In certain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 60, 70, 80, 90, 93, 95, 96, 97,98, or 99% of the aqueous phase by volume in droplet size between about15-45 μm, 20-40 μm, 20-45 μm, 25-40 μm, 20-35 μm, 30-40 μm, 35-40 μm, or25-45 μm, wherein percentage of the aqueous phase is based on the totalvolume of the aqueous phase. In certain embodiments, the mixing in stepa) creates an emulsion that comprises at least about 60, 70, 80, 90, 93,95, 96, 97, 98, or 99% of the aqueous phase by volume in droplet sizebetween about 15-45 μm, 20-40 μm, 20-45 μm, 25-40 μm, 20-35 μm, 30-40μm, 35-40 μm, or 25-45 μm, wherein percentage of the aqueous phase isbased on the total volume of the aqueous phase. In certain embodiments,the mixing in step b) creates an emulsion that comprises at least about60, 70, 80, 90, 93, 95, 96, 97, 98, or 99% of the aqueous phase byvolume in droplet size between about 15-45 μm, 20-40 μm, 20-45 μm, 25-40μm, 20-35 μm, 30-40 μm, 35-40 μm, or 25-45 μm, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase. Incertain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 10-30% of the aqueous phase byvolume in droplet size less than about 10 μm, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase. Incertain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 20-25% of the aqueous phase byvolume in droplet size less than about 10 μm, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase. Incertain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 60-95% of the aqueous phase byvolume in droplet size greater than about 10μ, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase. Incertain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 70-80% of the aqueous phase byvolume in droplet size greater than about 10μ, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase. Incertain embodiments, the mixing in steps a) and/or b) creates anemulsion that comprises at least about 90% of the aqueous phase byvolume in average droplet size of about 20μ, wherein percentage of theaqueous phase is based on the total volume of the aqueous phase.

Without being bound by any particular theory, it is believed that instep a), the calcium, magnesium, and iron salts of phosphatidic acid andphosphatidyl ethanolamine dissociate. The free calcium, magnesium, andiron cations react with, for example, citrate, acetate or phosphateanions from the acid, to form metal salts. In step b), the metal cationsfrom the base, for example sodium or potassium ions, form complexes withthe phosphatidic acid or the phosphatidyl ethanolamine. In the certainembodiments, the method further comprises addition of water followed bya high shear mixing to form a mechanical emulsion. The emulsifiedphospholipids are then removed by chemical degumming or reacted in theenzymatic degumming

Any shearing and/or mixing device deemed suitable by one of skill in theart can be used for mixing in the methods provided herein. In certainembodiments, mixing comprises shearing and agitation. In certainembodiment, the mixing device is an overhead mixer, including an IKA RW20 digital mixer with a flat blade paddle. In certain embodiments, themixing device is operated at about 50 rpm, 100 rpm, 150 rpm or 200 rpmfor normal agitation and about 250 rpm, 300 rpm, 350 rpm, 400 rpm ormore for vigorous agitation. In certain embodiment, the shear mixing isaccomplished with IKA's Ultra-Turrax homogenizer T-50 basic with a S 50N-G 45 G dispersion element at 10,000 rpm.

In certain embodiments, the mixer is a rotor/stator high shear mixerwith tip speed (radial velocity in the mixer chamber) of at least about1400 cm/s. In certain embodiments, and the power dissipated by the mixeris at least 1.0 KW/metric ton of product/h. In certain embodiments,oil/water emulsions in industrial scale are obtained with tip speedsranging from about 1400 cm/s to 2300 cm/s, or even higher. In certainembodiments, the tip speed is about 1400 cm/s, 1600 cm/s, 1800 cm/s,2000 cm/s, 2100 cm/s, 2300 cm/s, 2500 cm/s, 3000 cm/s, or 3500 cm/s. Incertain embodiments, the tip speed is about 2300 cm/s. In certainembodiments, and the power dissipated by the mixer is from about 1.0 toabout 2.0 KW/metric ton of product/h. In certain embodiments, and thepower dissipated by the mixer is about 2.0 KW/metric ton of product/h.In certain embodiments, for a continuous process, 10 KW of effectivepower dissipation in the high shear mixer is required for 10 metric tonof oil per hour.

In certain embodiments, mixing of acid comprises shearing for less thanabout 1 minute. In certain embodiments, mixing of acid comprisesshearing for about 1 second, 3 seconds, 5 seconds, 8 seconds, 10seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds or 60 seconds.In certain embodiments, mixing of acid comprises shearing for at leastabout 1 minute. In certain embodiments, mixing of acid comprisesshearing for at least about 1 second up to about 10 minutes, at leastabout 1 second up to about 10 minutes, at least about 1 second up toabout 7 minutes, at least about 1 second up to about 5 minutes, at leastabout 1 second up to about 3 minutes or at least about 1 second up toabout 2 minutes. In certain embodiments, mixing of acid comprisesshearing for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes.

In certain embodiments, mixing of acid comprises shearing followed byagitation from about 1 minute up to about 5 hours. In certainembodiments, the acidic mixture is agitated for at least about 1 minute.In certain embodiments, agitation of acid is continued from about 10minutes to about 5 hours or more. In certain embodiments, agitation ofacid is continued from about 30 minutes to about 5 hours or more. Incertain embodiments, agitation of acid is continued from about 30minutes to about 3 hours or more. In certain embodiments, agitation ofacid is continued from about 30 minute to about 2 hours or more. Incertain embodiments, agitation of acid is continued for about 1, 10, 20,30, 40, 50, 60, 70, 80, 90, 120, 150 or 180 minutes.

In certain embodiments, the acid used in step a) is selected from thegroup consisting of phosphoric acid, acetic acid, citric acid, tartaricacid, succinic acid, and mixtures thereof. In one embodiment, the acidis citric acid.

In certain embodiments, the pH of the acidic mixture in step a) is about1 to about 4. In certain embodiments, the pH of the acidic mixture instep a) is about 1, 1.5, 2, 2.5, 3, 3.5 or 4.

In certain embodiments, mixing of acid is continued till the calcium,magnesium, and iron salts of phosphatidic acid and phosphatidylethanolamine dissociate.

In certain embodiments, the aqueous acid used in the method comprises atleast about 5% by weight acid based on the combined weight of acid andwater. In certain embodiments, the aqueous acid used in the methodcomprises at least about 5 up to about 90%, about 5 up to about 80%,about 5 up to about 70%, about 10 up to about 90%, about 20 up to about60%, about 30 up to about 60%, or about 40 up to about 60% by weightacid based on the combined weight of acid and water. In certainembodiments, the aqueous acid used in the method comprises at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90% byweight acid based on the combined weight of acid and water. In certainembodiments, the aqueous acid used in the methods comprises at leastabout 5% by weight citric acid based on the combined weight of citricacid and water. In certain embodiments, the aqueous acid used in themethods comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55 or 60% by weight citric acid based on the combined weight of citricacid and water. In certain embodiments, the aqueous acid used in themethods comprises at least about 40, 45, 50, 55 or 60% by weight citricacid based on the combined weight of citric acid and water. In oneembodiment, the aqueous acid used in the method comprises about 50% byweight citric acid based on the combined weight of citric acid andwater. In certain embodiments, the aqueous acid used in the methodscomprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 85, 90% by weight phosphoric acid based on the combined weightof phosphoric acid and water.

In certain embodiments, the aqueous acid is used in at least about 0.01%by weight based on total weight of the oil. In certain embodiments, theaqueous acid is used in at least about 0.05% by weight based on totalweight of the oil. In certain embodiments, the aqueous acid is used inat least about 0.01 up to about 10%, about 0.01 up to about 5%, about0.05 up to about 5%, about 0.05 up to about 3%, about 0.05 up to about2% or about 0.1 up to about 2% by weight based on total weight of theoil. In certain embodiments, the aqueous acid is used in about 0.01,0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.5, 1, 2, 5, 7 or 10% by weight basedon total weight of the oil.

A base is mixed with the acidic mixture to obtain a reacted mixturehaving pH of about 6-9 at the aqueous phase. The mixing is continued toallow the non hydratable phospholipids within a lipid matrix to migrateto an oil-water interface. Any base deemed suitable by one of skill inthe art can be used in step b). In certain embodiments, the base isselected from the group consisting of sodium hydroxide, potassiumhydroxide, sodium silicate, sodium carbonate, calcium carbonate, and acombination thereof. In one embodiment, the base is sodium hydroxide. Incertain embodiments, the base is added as a dilute aqueous solution suchthat the base does not saponify any neutral oil. In certain embodiments,the base is added as about 0.1 up to about 8 M, about 1 up to about 4 M,about 1 up to about 3 M, or about 0.5 up to about 3 M aqueous solution.In certain embodiments, the base is added as about 0.1 M, 0.5 M, 1 M, 2M, 3 M, 4 M, 5 M, 6 M, 7 M or 8 M aqueous solution. In one embodiment,the minimum amount of base to be used for the removal of the NHPs to beeffective is such that the pH of the aqueous phase is raised to at leastabout 6. In certain embodiments, the amount of base used is sufficientto raise the pH of the aqueous phase to about 6, 6.5, 7, 7.5 or 8. Incertain embodiments, mixing of base is continued for at least about 1minute. In certain embodiments, mixing of base is continued from about 1minute to about 5 hours or more. In certain embodiments, mixing of baseis continued for about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150or 180 minutes.

The methods provided herein can be conducted at any temperature deemedsuitable by one of skill in the art. In certain embodiments, thetemperature during the process is in the range from about 20° C. toabout 100° C., about 20° C. to about 90° C., about 40° C. to about 80°C., or about 40° C. to about 70° C. In certain embodiments, thetemperature during the process is about 20, 30, 40, 50 60, 70, 80, 90 or100° C.

In certain embodiments, water is added after reaction with the base inan amount from about 0.1 to 5% or more based on the total volume of thereaction mixture followed by a high shear mixing to form a mechanicalemulsion enabling either phospholipids to be emulsified in the chemicaldegumming or reacted in the enzymatic degumming. In certain embodiments,water is then added in about 0.1, 0.5, 1, 2, 3, 4, 5% or more based onthe total volume of the reaction mixture.

In one embodiment, provided herein is a method wherein hydration of NHPsis followed by enzymatic treatment to remove various phospholipids andlecithins. Such methods can be practiced on either crude orwater-degummed oils.

In certain embodiments, an oil degumming method provided hereincomprises: a) mixing an aqueous acid with an edible oil to obtain anacidic mixture having pH of about 1 to 4; b) mixing a base with theacidic mixture to obtain a reacted mixture having pH of about 6-9; andc) degumming the reacted mixture with water or an enzyme to obtain adegummed oil, wherein the mixing in steps a) and/or b) is carried outwith a high shear mixer.

In the embodiments where an enzyme is used in the degumming step, one ormore enzymes can be added to the oil either separately or together.Enzymatic reaction parameters including temperature, pH, and enzymeconcentration can be controlled to optimize the reaction for aparticular enzyme combination in a particular oil system. Many varietiesof enzymes and their equivalents are suitable for use in the methodsprovided herein, including the phospholipase A and phospholipase Cfamilies that are available commercially. Exemplary enzymes aredescribed elsewhere herein.

In certain embodiments, the different phospholipases used together inthe enzymatic degumming step are mixed together before addition to theoil to be treated. Alternatively, the enzymes are added to the oilseparately, either sequentially or simultaneously.

The amount of enzyme used in the methods provided herein depends on thereaction conditions, the type of oil and the type of enzyme use. Incertain embodiments, the amount is in the range from 10 to 20,000 units,from 20 to 10,000 units, from 50 to 5,000 units, or from 100 to 2,000units, per 1 kg of the oil.

In one embodiment, provided herein is a method for removing NHPs,hydratable phospholipids, and lecithins (known collectively as “gums”)from vegetable oils to produce a degummed oil or fat product that can beused for food production and/or non-food applications. In certainembodiments, the degumming methods provided herein utilize water,various acids and/or various bases or a combination thereof.

In one embodiment, methods provided herein are useful for removal ofsalts of phosphatidic acid and phosphatidyl ethanolamine from vegetableoils. In certain embodiments, calcium and magnesium citrate salts areformed in step a). The methods provided herein eliminate the problemsassociated with equipment fouling due to deposition of calcium andmagnesium citrate salts on post-reaction equipments. The calcium andmagnesium citrate salts are soluble at the pH at which the enzymaticreaction and further processing is carried out in the methods providedherein.

In certain embodiments, provided herein are methods for enhancing thereaction rate of a phospholipase used in an enzymatic degumming, suchthat the enzyme reaction has a duration of less than about six, five,four, three, two or one hour. In certain embodiments, the enhancement inthe reaction rate is achieved by a high shear mixing of the reactedmixture of step b) to form a mechanical emulsion which is then reactedwith the enzyme.

It is yet another aspect, provides herein is a method for degumming avegetable oil composition in which both hydratable and non-hydratablephospholipids can be treated in a single process, wherein an enzymereaction is completed in less than about one hour.

In certain embodiment, the oil comprises Neochloris oleoabundans oil,Scenedesmus dimorphus oil, Euglena gracilis oil, Phaeodactylumtricornutum oil, Pleurochrysis carterae oil, Prymnesium parvum oil,Tetraselmis chui oil, Tetraselmis suecica oil, Isochrysis galbana oil,Nannochloropsis salina oil, Botryococcus braunii oil, Dunaliellatertiolecta oil, Nannochloris species oil, Spirulina species oil,Chlorophycease oil, Bacilliarophy oil, acai oil, almond oil, babassuoil, blackcurrent seed oil, borage seed oil, canola oil, cashew oil,castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambeoil, flax seed oil, grape seed oil, hazelnut oil, other nut oils,hempseed oil, jatropha oil, jojoba oil, linseed oil, macadamia nut oil,mango kernel oil, meadowfoam oil, mustard oil, neat's foot oil, oliveoil, palm oil, palm kernel oil, peanut oil, pecan oil, pine nut oil,pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, saffloweroil, sasanqua oil, sesame oil, shea butter oil, soybean oil, sunflowerseed oil, tall oil, tsubaki oil, walnut oil, varieties of “natural” oilshaving altered fatty acid compositions via Genetically ModifiedOrganisms (GMO) or traditional “breading” such as high oleic, lowlinolenic, or low saturated oils (high oleic canola oil, low linolenicsoybean oil or high stearic sunflower oils) or a blend of thereof. Inone embodiment, oils that can be treated include but are not limited tothe following: canola oil, castor oil, coconut oil, coriander oil, cornoil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, mangokernel oil, meadowfoam oil, neat's foot oil, olive oil, palm oil, palmkernel oil, palm olein, peanut oil, rapeseed oil, rice bran oil,safflower oil, sasanqua oil, soybean oil, sunflower seed oil, tall oil,tsubaki oil, and vegetable oil.

In certain embodiments, the methods provided herein reduce thephospholipids content of an oil to less than about 30 ppm phosphorus,less than about 20 ppm phosphorus, less than about 15 ppm phosphorus,less than about 10 ppm phosphorus, less than about 7 ppm phosphorus,less than about 5 ppm phosphorus or less than about 3 ppm phosphorus. Incertain embodiments, the methods provided herein reduce the phospholipidcontent of an oil to about 10 ppm phosphorus, about 7 ppm phosphorus,about 5 ppm phosphorus or about 3 ppm phosphorus.

After the degumming step, the degummed oil can be separated from thegums, and subjected to further processing steps known in the artincluding bleaching or deodorizing, as may be necessary or desirabledepending on the end use for which the degummed oil product is intended.

In certain embodiment, provided herein are methods for obtainingphospholipids comprising:

-   -   a) mixing an aqueous acid with the edible oil to obtain an        acidic mixture having pH of less than about 4;    -   b) mixing a base with the acidic mixture to obtain a reacted        mixture having pH of about 6-9, wherein the mixing in steps a)        and/or b) creates an emulsion that comprises at least about 60%        of an aqueous phase by volume in droplet size between about 15        μm to about 45 μm in size;    -   c) mixing an enzyme selected from phospholipase A, phospholipase        C, phosphatidyl-inositol specific phospholipase C, or a        combination thereof; and    -   d) isolating the phospholipids.

In various exemplary processes of the invention phosphorus levels arereduced low enough for physical refining. The separation process canresult in potentially higher yield losses than caustic refining.Additionally, degumming processes may generate waste products that maynot be sold as commercial lecithin, see, e.g., FIG. 2 for an exemplarydegumming process for physically refined oils. Therefore, theseprocesses have not achieved a significant share of the market andcaustic refining processes continue to dominate the industry for ricebran, soy, canola and sunflower. Note however, that a phospholipase Cenzyme employed in a special degumming process would decrease gumformation and return the diglyceride portion of the phospholipid back tothe oil.

In one aspect, the invention provides methods using a PI-PLC of theinvention in the gum fraction. In one aspect of this method, oil isadded to the crude oil to create an emulsion that results in themovement of the phosphatidylcholine, phosphatidyl-ethanolamine andphosphatidylinositol into the aqueous phase (water degumming) Followingcentrifugation, these phospholipids are major components of the aqueousgum fraction. The phospholipids in the gum fraction can be treated withphospholipase C or phospholipase D plus phosphatase (or othercombinations, noted below) to generate diacylglycerol (DAG) and aphosphate ester. At this point, the DAG can be extracted from the othergum components and treated with a lipase under conditions suitable forthe transesterification of the DAG to produce a desired triacylglycerol(structured lipid).

In another aspect, the majority of the 1,2-DAG can be converted to1,3-DAG by increasing the pH of the gum following the PLC reaction, forexample, by adding caustic. The 1,3-DAG can then be extracted from thegum and reacted with a lipase under the appropriate conditions totransesterify the 1,3-DAG at the sn2 position to create the desiredstructured triacylglycerol.

In alternative aspects, the fatty acids used in the transesterificationreaction could come from a variety of sources including the free fattyacids found in the crude oil.

In one aspect, the phospholipids from water degumming are used incombination with a PLC of the invention to create structured lipids. Thewater-degummed oil can be exposed to a PLC and/or PLD (either or bothcan be enzymes of the invention) plus phosphatase or one of thesecombinations followed by PLA (can be an enzyme of the invention) toreduce the phosphorus to levels suitable for caustic or physicalrefining.

In alternative embodiments, combination of enzymes that can be used inthe methods of the invention, including these degumming processes,include (where one, several or all of the enzymes in the combinationcomprise an enzyme of the instant invention):

-   -   PLC+PI-PLC+PLA (PLA added after completion of PLC reactions);    -   PLD+phosphatase+PI-PLC followed by PLA; or,    -   PLC or (PLC+PI-PLC)+PLA specific for phosphatidic acid (all        enzymes added together or sequentially).

Caustic Refining

The invention provides processes using phospholipases (including enzymesof the invention) in caustic refining, where the enzymes are used ascaustic refining aids. In alternative aspects, a PLC or PLD and/or aphosphatase are used in the processes as adrop-in, either before,during, or after a caustic neutralization refining process (eithercontinuous or batch refining). The amount of enzyme added may varyaccording to the process. The water level used in the process can below, e.g., about 0.5 to 5%. Alternatively, caustic is be added to theprocess multiple times. In addition, the process may be performed atdifferent temperatures (25° C. to 70° C.), with different acidsorcaustics, and at varying pH (4-12). Concentrated solutions of caustic,e.g., more concentrated than the industrial standard of 11%, to decreasemass of gum can be used. In alternative aspects, the concentratedsolution of caustic is between about 12% and 50% concentrated, e.g.,about 20%, 30%, 40%, 50%, or 60% or more concentrated.

In one aspect, a phospholipase C enzyme of the invention hydrolyzes aphosphatide at a glyceryl phosphoester bond to generate a diglycerideand water-soluble phosphate compound. The hydrolyzed phosphatide movesto the aqueous phase, leaving the diglyceride in the oil phase, asillustrated in FIG. 3. One objective of the PLC “Caustic Refining Aid”is to convert the phospholipid gums formed during neutralization into adiacylglyceride that will migrate back into the oil phase. In contrast,one objective of the “PLC Degumming Aid” is to reduce the phospholipidsin crude oil to a phosphorus equivalent of less than 10 parts permillion (ppm).

Acids that may be used in a caustic refining process include, but arenot limited to, phosphoric, citric, ascorbic, sulfuric, fumaric, maleic,hydrochloric and/or acetic acids. Acids are used to hydratenon-hydratable phospholipids. Caustics that may be used include, but arenot limited to, KOH and NaOH. Caustics are used to neutralize free fattyacids. Alternatively, phospholipases, or more particularly a PLC or aPLD and a phosphatase, are used for purification of phytosterols fromthe gum/soapstock.

An alternate embodiment of the invention to add the phospholipase beforecaustic refining is to express the phospholipase in a plant. In anotherembodiment, the phospholipase is added during crushing of the plant,seeds or other plant part. Alternatively, the phospholipase is addedfollowing crushing, but prior to refining (i.e. in holding vessels). Inaddition, phospholipase is added as a refining pre-treatment, eitherwith or without acid.

Another embodiment of the invention, already described, is to add thephospholipase during a caustic refining process. In this process, thelevels of acid and caustic are varied depending on the level ofphosphorus and the level of free fatty acids. In addition, broadtemperature and pH ranges are used in the process, dependent upon thetype of enzyme used.

In another embodiment of the invention, the phospholipase is added aftercaustic refining (FIG. 5). In one instance, the phospholipase is addedin an intense mixer or in a retention mixer, prior to separation.Alternatively, the phospholipase is added following the heat step. Inanother embodiment, the phospholipase is added in the centrifugationstep. In an additional embodiment, the phospholipase is added to thesoapstock. Alternatively, the phospholipase is added to the washwater.In another instance, the phospholipase is added during the bleachingand/or deodorizing steps.

In one aspect, a phospholipase, e.g., a phospholipase C, enzyme of theinvention will hydrolyze the phosphatide from both hydratable andnon-hydratable phospholipids in neutralized crude and degummed oilsbefore bleaching and deodorizing. Exemplary “caustic refining” processesof the invention are illustrated in FIG. 4 and FIG. 6. FIG. 4 includesexemplary times, temperature and pHs for static mixer (30 to 60 min, 50to 60° C., pH 5 to 6.5) and retention mixer (3 to 10 min, 20 to 40° C.).The target enzyme can be applied as a drop-in product in the existingcaustic neutralization process, as illustrated in FIG. 4. In thisaspect, the enzyme will not be required to withstand extreme pH levelsif it is added after the addition of caustic. As illustrated in FIG. 6(an enzyme “front loading” exemplary process), any phospholipase,including, e.g., a phospholipase of the invention, such as a PLC,PI-PLC, PLB, PLA and/or PLC, can be used in a crude oil degummingprocess, as described, e.g., in Bailey's Industrial Oil & Fat Productsv.4 (ed. Y. H. Hui). FIG. 7 and FIG. 8 illustrate variations of methodsof the invention where two or three centrifugation steps, respectively,are used in the process, which can be used to process any oil, e.g., avegetable oil such as crude soy oil, as shown in the figure. Theexemplary method of FIG. 8 has a centrifugation step before causticrefining (in addition to a centrifugation step after caustic refiningand before the water wash, and, after the water wash), while theexemplary method of FIG. 7 does not have a centrifugation step beforecaustic refining. FIG. 9 illustrates another exemplary variation of thisprocess using acid treatment and having a centrifugation step before adegumming step; this exemplary process can be used to process any oil,e.g., a vegetable oil such as crude soy oil, as shown in the figure.

In one aspect, a phospholipase of the invention enables phosphorus to beremoved to the low levels acceptable in physical refining. In oneaspect, a PLC of the invention will hydrolyze the phosphatide from bothhydratable and non-hydratable phospholipids in crude oils beforebleaching and deodorizing. The target enzyme can be applied as a drop-inproduct in an existing degumming operation, see, e.g., FIG. 5. Givensub-optimal mixing in commercial equipment, it is likely that acid willbe required to bring the non-hydratable phospholipids in contact withthe enzyme at the oil/water interface. Therefore, in one aspect, anacid-stable PLC of the invention is used.

In one aspect, a PLC Degumming Aid process of the invention caneliminate losses in one, or all three, areas noted in Table 4. Lossesassociated in a PLC process can be estimated to be about 0.8% versus5.2% on a mass basis due to removal of the phosphatide.

TABLE 4 Losses Addressed by PLC Products Caustic Refining Degumming AidAid 1) Oil lost in gum formation & 2.1% X X separation 2) Saponified oilin caustic addition 3.1% X 3) Oil trapped in clay in bleaching* <1.0% XX Total Yield Loss ~5.2% ~2.1% ~5.2%

Additional potential benefits of this process of the invention includethe following:

-   -   Reduced adsorbents—less adsorbents required with lower (<5 ppm)        phosphorus    -   Lower chemical usage—less chemical and processing costs        associated with hydration of non-hydratable phospholipids    -   Lower waste generation—less water required to remove phosphorus        from oil

Oils processed (e.g., “degummed”) by the methods of the inventioninclude plant oilseeds, e.g., soybean oil, rapeseed oil, rice bran oiland sunflower oil. In one aspect, the “PLC Caustic Refining Aid” of theinvention can save 1.2% over existing caustic refining processes. Therefining aid application addresses soy oil that has been degummed forlecithin and these are also excluded from the value/load calculations.

Performance targets of the processes of the invention can vary accordingto the applications and more specifically to the point of enzymeaddition, see Table 5.

TABLE 5 Performance Targets by Application Caustic Degumming RefiningAid Aid Incoming Oil Phosphorus Levels <200 ppm* 600-1,400 ppm Final OilPhosphorus Levels <10 ppm^(†) <10 ppm Hydratable & Non-hydratable gumsYes Yes Residence Time 3-10 minutes 30 minutes^(‡) Liquid FormulationYes Yes Target pH 8-10^(‡‡‡) 5.0-5.5^(‡‡) Target Temperature 20-40° C.~50-60° C. Water Content <5% 1-1.25% Enzyme Formulation Purity Nolipase/ No lipase/ protease protease Other Key Requirements Removal ofFe Removal of Fe *Water degummed oil ^(†)Target levels achieved inupstream caustic neutralization step but must be maintained ^(‡)1-2hours existing ^(‡‡)Acid degumming will require an enzyme that is stablein much more acidic conditions: pH at 2.3 for citric acid at 5%. (~RoehmUSPN 6,001,640). ^(‡‡‡)The pH of neutralized oil is NOT neutral. Testingat POS indicates that the pH will be in the alkaline range from 6.5-10(Dec. 9, 2002). Typical pH range needs to be determined.

Other processes that can be used with a phospholipase of the invention,e.g., a phospholipase A₁ can convert non-hydratable native phospholipidsto a hydratable form. In one aspect, the enzyme is sensitive to heat.This may be desirable, since heating the oil can destroy the enzyme.However, the degumming reaction must be adjusted to pH 4-5 and 60° C. toaccommodate this enzyme. At 300 Units/kg oil saturation dosage, thisexemplary process is successful at taking previously water-degummed oilphosphorus content down to ≦10 ppm P. Advantages can be decreased H₂Ocontent and resultant savings in usage, handling and waste. Table 6lists exemplary applications for industrial uses for enzymes of theinvention:

TABLE 6 Exemplary Application Caustic Refining Degumming Aid Aid Soy oilw/ lecithin production X Chemical refined soy oil, Sunflower oil, X XCanola oil Low phosphatide oils (e.g. palm) X

In addition to these various “degumming” processes, the phospholipasesof the invention can be used in any vegetable oil processing step. Forexample, phospholipase enzymes of the invention can be used in place ofPLA, e.g., phospholipase A2, in any vegetable oil processing step. Oilsthat are “processed” or “degummed” in the methods of the inventioninclude soybean oils, rapeseed oils, corn oils, oil from palm kernels,canola oils, sunflower oils, sesame oils, peanut oils, rice bran oil andthe like. The main products from this process include triglycerides.

In one exemplary process, when the enzyme is added to and reacted with acrude oil, the amount of phospholipase employed is about 10-10,000units, or, alternatively, about, 100-2,000 units, per 1 kg of crude oil.The enzyme treatment is conducted for 5 min to 10 hours at a temperatureof 30° C. to 90° C., or, alternatively, about, 40° C. to 70° C. Theconditions may vary depending on the optimum temperature of the enzyme.The amount of water added to dissolve the enzyme is 5-1,000 wt. partsper 100 wt. parts of crude oil, or, alternatively, about, 10 to 200 wt.parts per 100 wt. parts of crude oil.

Upon completion of such enzyme treatment, the enzyme liquid is separatedwith an appropriate means such as a centrifugal separator and theprocessed oil is obtained. Phosphorus-containing compounds produced byenzyme decomposition of gummy substances in such a process arepractically all transferred into the aqueous phase and removed from theoil phase. Upon completion of the enzyme treatment, if necessary, theprocessed oil can be additionally washed with water or organic orinorganic acid such as, e.g., acetic acid, citric acid, phosphoric acid,succinic acid, and equivalent acids, or with salt solutions.

In one exemplary process for ultra-filtration degumming, the enzyme isbound to a filter or the enzyme is added to an oil prior to filtrationor the enzyme is used to periodically clean filters.

In one exemplary process for a phospholipase-mediated physical refiningaid, water and enzyme are added to crude oil (e.g., crude vegetableoil). In one aspect, a PLC or a PLD of the invention and a phosphataseare used in the process. In phospholipase-mediated physical refining,the water level can be low, i.e. 0.5-5% and the process time should beshort (less than 2 hours, or, less than 60 minutes, or, less than 30minutes, or, less than 15 minutes, or, less than 5 minutes). The processcan be run at different temperatures (25° C. to 70° C.), using differentacids and/or caustics, at different pHs (e.g., 3-10).

In alternate aspects, water degumming is performed first to collectlecithin by centrifugation and then PLC or PLC and PLA of the inventionis added to remove non-hydratable phospholipids (the process should beperformed under low water concentration). In another aspect, waterdegumming of crude oil to less than 10 ppm (edible oils) and subsequentphysical refining (less than 50 ppm for biodiesel) is performed. In oneaspect, an emulsifier is added and/or the crude oil is subjected to anintense mixer to promote mixing. Alternatively, an emulsion-breaker isadded and/or the crude oil is heated to promote separation of theaqueous phase. In another aspect, an acid is added to promote hydrationof non-hydratable phospholipids. Additionally, phospholipases can beused to mediate purification of phytosterols from the gum/soapstock.

In one aspect, the invention provides compositions and methods (whichcan comprise use of phospholipases of the invention) for oil degummingcomprising using varying amounts of acid and base without makingsoapstock. Using this aspect of the invention for oil degumming, acid(including phosphoric and/or citric) can be used to hydratenon-hydratable phospholipids in high phosphorus oils (including soybean,canola, and sunflower). Once the phospholipids are hydrated, the pH ofthe aqueous phase can be raised using caustic addition: the amount ofcaustic added can create a favorable pH for enzyme activity but will notresult in the formation of a significant soapstock fraction in the oil.Because a soapstock is not formed, the free fatty acids in the oil canbe removed downstream, following the degumming step, during bleachingand deodorization.

Enzymes of the invention are used to improve oil extraction and oildegumming (e.g., vegetable oils). In one aspect, a PLC of the inventionand at least one plant cell wall degrader (e.g., a cellulase, ahemicellulase or the like, to soften walls and increase yield atextraction) is used in a process of the invention. In this exemplaryapproach to using enzymes of the invention to improve oil extraction andoil degumming, a phospholipase C of the invention as well as otherhydrolases (e.g., a cellulase, a hemicellulase, an esterase, a proteaseand/or a phosphatase) are used during the crushing steps associated withoil production (including but not limited to soybean, canola, sunflower,rice bran oil). By using enzymes prior to or in place of solventextraction, it is possible to increase oil yield and reduce the amountof hydratable and non-hydratable phospholipids in the crude oil. Thereduction in non-hydratable phospholipids may result from conversion ofpotentially non-hydratable phospholipids to diacylglycerol andcorresponding phosphate-ester prior to complexation with calcium ormagnesium. The overall reduction of phospholipids in the crude oil willresult in improved yields during refining with the potential foreliminating the requirement for a separate degumming step prior tobleaching and deodorization.

In one aspect, the invention provides processes using a phospholipase ofthe invention (e.g., a phospholipase-specific phosphohydrolase of theinvention), or another phospholipase, in a modified “organic refiningprocess,” which can comprise addition of the enzyme (e.g., a PI-PLC) ina citric acid holding tank.

The enzymes of the invention can be used in any oil processing method,e.g., degumming or equivalent processes. For example, the enzymes of theinvention can be used in processes as described in U.S. Pat. Nos.5,558,781; 5,264,367; 6,001,640. The process described in U.S. Pat. No.5,558,781 uses either phospholipase A1, A2 or B, essentially breakingdown lecithin in the oil that behaves as an emulsifier.

The enzymes and methods of the invention can be used in processes forthe reduction of phosphorus-containing components in edible oilscomprising a high amount of non-hydratable phosphorus by using of aphospholipase of the invention, e.g., a polypeptide having aphospholipase A and/or B activity, as described, e.g., in EP PatentNumber: EP 0869167. In one aspect, the edible oil is a crude oil, aso-called “non-degummed oil.” In one aspect, the method treat anon-degummed oil, including pressed oils or extracted oils, or a mixturethereof, from, e.g., rapeseed, soybean, sesame, peanut, corn, rice branor sunflower. The phosphatide content in a crude oil can vary from 0.5to 3% w/w corresponding to a phosphorus content in the range of 200 to1200 ppm, or, in the range of 250 to 1200 ppm. Apart from thephosphatides, the crude oil can also contain small concentrations ofcarbohydrates, sugar compounds and metal/phosphatide acid complexes ofCa, Mg and Fe. In one aspect, the process comprises treatment of aphospholipid or lysophospholipid with the phospholipase of the inventionso as to hydrolyze fatty acyl groups. In one aspect, the phospholipid orlysophospholipid comprises lecithin or lysolecithin. In one aspect ofthe process the edible oil has a phosphorus content from between about50 to 250 ppm, and the process comprises treating the oil with aphospholipase of the invention so as to hydrolyze a major part of thephospholipid and separating an aqueous phase containing the hydrolyzedphospholipid from the oil. In one aspect, prior to the enzymaticdegumming process the oil is water-degummed. In one aspect, the methodsprovide for the production of an animal feed comprising mixing thephospholipase of the invention with feed substances and at least onephospholipid.

The enzymes and methods of the invention can be used in processes of oildegumming as described, e.g., in WO 98/18912. The phospholipases of theinvention can be used to reduce the content of phospholipid in an edibleoil. The process can comprise treating the oil with a phospholipase ofthe invention to hydrolyze a major part of the phospholipid andseparating an aqueous phase containing the hydrolyzed phospholipid fromthe oil. This process is applicable to the purification of any edibleoil, which contains a phospholipid, e.g. vegetable oils, such as soybeanoil, rice bran oil, rapeseed oil and sunflower oil, fish oils, algae andanimal oils and the like. Prior to the enzymatic treatment, thevegetable oil is preferably pretreated to remove slime (mucilage), e.g.by wet refining. The oil can contain between about 50 to 250 ppm, orbetween 50 to about 1500 ppm, or more, of phosphorus, as phospholipid atthe start of the treatment with phospholipase, and the process of theinvention can reduce this value to below between about 5 to 10 ppm.

The enzymes of the invention can be used in processes as described in JPApplication No.: H5-132283, filed Apr. 25, 1993, which comprises aprocess for the purification of oils and fats comprising a step ofconverting phospholipids present in the oils and fats into water-solublesubstances containing phosphoric acid groups and removing them aswater-soluble substances. An enzyme action is used for the conversioninto water-soluble substances. An enzyme having a phospholipase Cactivity is preferably used as the enzyme.

The enzymes of the invention can be used in processes as described asthe “Organic Refining Process,” (ORP) (IPH, Omaha, Nebr.) which is amethod of refining seed oils. ORP may have advantages over traditionalchemical refining, including improved refined oil yield, value addedco-products, reduced capital costs and lower environmental costs.

The enzymes of the invention can be used in processes for the treatmentof an oil or fat, animal or vegetal, raw, semi-processed or refined,comprising adding to such oil or fat at least one enzyme of theinvention that allows hydrolyzing and/or depolymerizing thenon-glyceridic compounds contained in the oil, as described, e.g., in EPApplication number: 82870032.8. Exemplary methods of the invention forhydrolysis and/or depolymerization of non-glyceridic compounds in oilsare:

-   1) The addition and mixture in oils and fats of an enzyme of the    invention or enzyme complexes previously dissolved in a small    quantity of appropriate solvent (for example water). A certain    number of solvents are possible, but a non-toxic and suitable    solvent for the enzyme is chosen. This addition may be done in    processes with successive loads, as well as in continuous processes.    The quantity of enzyme(s) necessary to be added to oils and fats,    according to this process, may range, depending on the enzymes and    the products to be processed, from between about 5 to 400 ppm, or    between about 20 to 400 ppm; e.g., 0.005 kg to 0.4 kg of enzyme for    1000 kg of oil or fat, and preferably from 5 to 100 ppm, i.e., from    0.005 to 0.1 kg of enzyme for 1000 kg of oil, these values being    understood to be for concentrated enzymes, i.e., without diluent or    solvent.-   2) Passage of the oil or fat through a fixed or insoluble filtering    bed of enzyme(s) of the invention on solid or semi-solid supports,    preferably presenting a porous or fibrous structure. In this    technique, the enzymes are trapped in the micro-cavities of the    porous or fibrous structure of the supports. These consist, for    example, of resins or synthetic polymers, cellulose carbonates, gels    such as agarose, filaments of polymers or copolymers with porous    structure, trapping small droplets of enzyme in solution in their    cavities. Concerning the enzyme concentration, it is possible to go    up to the saturation of the supports.-   3) Dispersion of the oils and fats in the form of fine droplets, in    a diluted enzymatic solution, in alternative aspects containing    between about 0.05 to 4%, or containing between about 0.2 to 4%, in    volume of an enzyme of the invention. This technique is described,    e.g., in Belgian patent No. 595,219. A cylindrical column with a    height of several meters, with conical lid, is filled with a diluted    enzymatic solution. For this purpose, a solvent that is non-toxic    and non-miscible in the oil or fat to be processed, preferably    water, is chosen. The bottom of the column is equipped with a    distribution system in which the oil or fat is continuously injected    in an extremely divided form (approximately 10,000 flux per m²).    Thus an infinite number of droplets of oil or fat are formed, which    slowly rise in the solution of enzymes and meet at the surface, to    be evacuated continuously at the top of the conical lid of the    reactor.

Palm oil can be pre-treated before treatment with an enzyme of theinvention. For example, about 30 kg of raw palm oil is heated to +50° C.1% solutions were prepared in distilled water with cellulases andpectinases. 600 g of each of these was added to aqueous solutions of theoil under strong agitation for a few minutes. The oil is then kept at+50° C. under moderate agitation, for a total reaction time of twohours. Then, temperature is raised to +90° C. to deactivate the enzymesand prepare the mixture for filtration and further processing. The oilis dried under vacuum and filtered with a filtering aid.

The enzymes of the invention can be used in processes as described in EPpatent EP 0 513 709 B2. For example, the invention provides a processfor the reduction of the content process for the reduction of thecontent of phosphorus-containing components in animal and vegetable oilsby enzymatic decomposition using a phospholipase of the invention. Inalternative aspects, predemucilaginated animal and vegetable oil with aphosphorus content of between about of 50 to 1500 ppm, or, between about50 to 250 ppm, is agitated with an organic carboxylic acid and the pHvalue of the resulting mixture set to between about pH 4 to pH 6, anenzyme solution which contains phospholipase A₁, A₂, or B of theinvention is added to the mixture in a mixing vessel under turbulentstirring and with the formation of fine droplets, where an emulsion with0.5 to 5% by weight relative to the oil is formed, said emulsion beingconducted through at least one subsequent reaction vessel underturbulent motion during a reaction time of 0.1 to 10 hours attemperatures in the range of 20 to 80° C. and where the treated oil,after separation of the aqueous solution, has a phosphorus content under5 ppm.

The organic refining process is applicable to both crude and degummedoil. The process uses inline addition of an organic acid undercontrolled process conditions, in conjunction with conventionalcentrifugal separation. The water separated naturally from the vegetableoil phospholipids (“VOP”) is recycled and reused. The total water usagecan be substantially reduced as a result of the Organic RefiningProcess.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,162,623. In this exemplary methods, the invention provides anamphiphilic enzyme. It can be immobilized, e.g., by preparing anemulsion containing a continuous hydrophobic phase and a dispersedaqueous phase containing the enzyme and a carrier for the enzyme andremoving water from the dispersed phase until this phase turns intosolid enzyme coated particles. The enzyme can be a lipase. Theimmobilized lipase can be used for reactions catalyzed by lipase such asinteresterification of mono-, di- or triglycerides, de-acidification ofa triglyceride oil, or removal of phospholipids from a triglyceride oilwhen the lipase is a phospholipase. The aqueous phase may contain afermentation liquid, an edible triglyceride oil may be the hydrophobicphase, and carriers include sugars, starch, dextran, water solublecellulose derivatives and fermentation residues. This exemplary methodcan be used to process triglycerides, diglycerides, monoglycerides,glycerol, phospholipids, glycolipids or fatty acids, which may be in thehydrophobic phase. In one aspect, the process for the removal ofphospholipids from triglyceride oil comprising mixing a triglyceride oilcontaining phospholipids with a preparation containing a phospholipaseof the invention; hydrolyzing the phospholipids to lysophospholipid;separating the hydrolyzed phospholipids from the oil, wherein thephospholipase is an immobilized phospholipase.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,127,137. This exemplary method hydrolyzes both fatty acyl groups inintact phospholipid. The phospholipase of the invention used in thisexemplary method has no lipase activity and is active at very low pH.These properties make it very suitable for use in oil degumming, asenzymatic and alkaline hydrolysis (saponification) of the oil can bothbe suppressed. In one aspect, the invention provides a process forhydrolyzing fatty acyl groups in a phospholipid or lysophospholipidcomprising treating the phospholipid or lysophospholipid with thephospholipase that hydrolyzes both fatty acyl groups in a phospholipidand is essentially free of lipase activity. In one aspect, thephospholipase of the invention has a temperature optimum at about 50°C., measured at pH 3 to pH 4 for 10 minutes, and a pH optimum of aboutpH 3, measured at 40° C. for about 10 minutes. In one aspect, thephospholipid or lysophospholipid comprises lecithin or lysolecithin. Inone aspect, after hydrolyzing a major part of the phospholipid, anaqueous phase containing the hydrolyzed phospholipid is separated fromthe oil. In one aspect, the invention provides a process for removingphospholipid from an edible oil, comprising treating the oil at pH 1.5to 3 with a dispersion of an aqueous solution of the phospholipase ofthe invention, and separating an aqueous phase containing the hydrolyzedphospholipid from the oil. In one aspect, the oil is treated to removemucilage prior to the treatment with the phospholipase. In one aspect,the oil prior to the treatment with the phospholipase contains thephospholipid in an amount corresponding to 50 to 250 ppm of phosphorus.In one aspect, the treatment with phospholipase is done at 30° C. to 45°C. for 1 to 12 hours at a phospholipase dosage of 0.1 to 10 mg/l in thepresence of 0.5 to 5% of water.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,025,171. In this exemplary methods, enzymes of the invention areimmobilized by preparing an emulsion containing a continuous hydrophobicphase, such as a triglyceride oil, and a dispersed aqueous phasecontaining an amphiphilic enzyme, such as lipase or a phospholipase ofthe invention, and carrier material that is partly dissolved and partlyundissolved in the aqueous phase, and removing water from the aqueousphase until the phase turns into solid enzyme coated carrier particles.The undissolved part of the carrier material may be a material that isinsoluble in water and oil, or a water soluble material in undissolvedform because the aqueous phase is already saturated with the watersoluble material. The aqueous phase may be formed with a crude lipasefermentation liquid containing fermentation residues and biomass thatcan serve as carrier materials. Immobilized lipase is useful for esterre-arrangement and de-acidification in oils. After a reaction, theimmobilized enzyme can be regenerated for a subsequent reaction byadding water to obtain partial dissolution of the carrier, and with theresultant enzyme and carrier-containing aqueous phase dispersed in ahydrophobic phase evaporating water to again form enzyme coated carrierparticles.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,143,545. This exemplary method is used for reducing the content ofphosphorus containing components in an edible oil comprising a highamount of non-hydratable phosphorus content using a phospholipase of theinvention. In one aspect, the method is used to reduce the content ofphosphorus containing components in an edible oil having anon-hydratable phosphorus content of at least 50 ppm measured bypre-treating the edible oil, at 60° C., by addition of a solutioncomprising citric acid monohydrate in water (added water vs. oil equals4.8% w/w; (citric acid) in water phase=106 mM, in water/oil emulsion=4.6mM) for 30 minutes; transferring 10 ml of the pre-treated water in oilemulsion to a tube; heating the emulsion in a boiling water bath for 30minutes; centrifuging at 5000 rpm for 10 minutes, transferring about 8ml of the upper (oil) phase to a new tube and leaving it to settle for24 hours; and drawing 2 g from the upper clear phase for measurement ofthe non-hydratable phosphorus content (ppm) in the edible oil. Themethod also can comprise contacting an oil at a pH from about pH 5 to 8with an aqueous solution of a phospholipase A or B of the invention(e.g., PLA1, PLA2, or a PLB), which solution is emulsified in the oiluntil the phosphorus content of the oil is reduced to less than 11 ppm,and then separating the aqueous phase from the treated oil.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.5,532,163. The invention provides processes for the refining of oil andfat by which phospholipids in the oil and fat to be treated can bedecomposed and removed efficiently. In one aspect, the inventionprovides a process for the refining of oil and fat which comprisesreacting, in an emulsion, the oil and fat with an enzyme of theinvention, e.g., an enzyme having an activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids (e.g., a PLA2 ofthe invention); and another process in which the enzyme-treated oil andfat is washed with water or an acidic aqueous solution. In one aspect,the acidic aqueous solution to be used in the washing step is a solutionof at least one acid, e.g., citric acid, acetic acid, phosphoric acidand salts thereof. In one aspect, the emulsified condition is formedusing 30 weight parts or more of water per 100 weight parts of the oiland fat. Since oil and fat can be purified without employing theconventional alkali refining step, generation of washing waste water andindustrial waste can be reduced. In addition, the recovery yield of oilis improved because loss of neutral oil and fat due to their inclusionin these wastes does not occur in the inventive process. In one aspect,the invention provides a process for refining oil and fat containingabout 100 to 10,000 ppm of phospholipids which comprises: reacting, inan emulsified condition, said oil and fat with an enzyme of theinvention having activity to decompose glycerol-fatty acid ester bondsin glycerophospholipids. In one aspect, the invention provides processesfor refining oil and fat containing about 100 to 10,000 ppm ofphospholipids which comprises reacting, in an emulsified condition, oiland fat with an enzyme of the invention having activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids; andsubsequently washing the treated oil and fat with a washing water.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.5,264,367. The content of phosphorus-containing components and the ironcontent of an edible vegetable or animal oil, such as an oil, e.g.,soybean oil, which has been wet-refined to remove mucilage, are reducedby enzymatic decomposition by contacting the oil with an aqueoussolution of an enzyme of the invention, e.g., a phospholipase A1, A2, orB, and then separating the aqueous phase from the treated oil. In oneaspect, the invention provides an enzymatic method for decreasing thecontent of phosphorus- and iron-containing components in oils, whichhave been refined to remove mucilage. An oil, which has been refined toremove mucilage, can be treated with an enzyme of the invention, e.g.,phospholipase C, A1, A2, or B. Phosphorus contents below 5 ppm and ironcontents below 1 ppm can be achieved. The low iron content can beadvantageous for the stability of the oil.

The phospholipases and methods of the invention can also be used forpreparing transesterified oils, as described, e.g., in U.S. Pat. No.5,288,619. The invention provides methods for enzymatictransesterification for preparing a margarine oil having both lowtrans-acid and low intermediate chain fatty acid content. The methodincludes the steps of providing a transesterification reaction mixturecontaining a stearic acid source material and an edible liquid vegetableoil, transesterifying the stearic acid source material and the vegetableoil using a 1-, 3-positionally specific lipase, and then finallyhydrogenating the fatty acid mixture to provide a recycle stearic acidsource material for a recyclic reaction with the vegetable oil. Theinvention also provides a counter-current method for preparing atransesterified oil. The method includes the steps of providing atransesterification reaction zone containing a 1-, 3-positionallyspecific lipase, introducing a vegetable oil into thetransesterification zone, introducing a stearic acid source material,conducting a supercritical gas or subcritical liquefied gascounter-current fluid, carrying out a transesterification reaction ofthe triglyceride stream with the stearic acid or stearic acid monoesterstream in the reaction zone, withdrawing a transesterified triglyceridemargarine oil stream, withdrawing a counter-current fluid phase,hydrogenating the transesterified stearic acid or stearic acid monoesterto provide a hydrogenated recycle stearic acid source material, andintroducing the hydrogenated recycle stearic acid source material intothe reaction zone.

In one aspect, the highly unsaturated phospholipid compound may beconverted into a triglyceride by appropriate use of a phospholipase C ofthe invention to remove the phosphate group in the sn-3 position,followed by 1,3 lipase acyl ester synthesis. The 2-substitutedphospholipid may be used as a functional food ingredient directly, ormay be subsequently selectively hydrolyzed in reactor 160 using animmobilized phospholipase C of the invention to produce a 1-diglyceride,followed by enzymatic esterification as described herein to produce atriglyceride product having a 2-substituted polyunsaturated fatty acidcomponent.

The phospholipases and methods of the invention can also be used in avegetable oil enzymatic degumming process as described, e.g., in U.S.Pat. No. 6,001,640. This method of the invention comprises a degummingstep in the production of edible oils. Vegetable oils from whichhydratable phosphatides have been eliminated by a previous aqueousdegumming process are freed from non-hydratable phosphatides byenzymatic treatment using a phospholipase of the invention. The processcan be gentle, economical and environment-friendly. Phospholipases thatonly hydrolyze lysolecithin, but not lecithin, are used in thisdegumming process.

In one aspect, to allow the enzyme of the invention to act, both phases,the oil phase and the aqueous phase that contain the enzyme, must beintimately mixed. It may not be sufficient to merely stir them. Gooddispersion of the enzyme in the oil is aided if it is dissolved in asmall amount of water, e.g., 0.5-5 weight-% (relative to the oil), andemulsified in the oil in this form, to form droplets of less than 10micrometers in diameter (weight average). The droplets can be smallerthan 1 micrometer. Turbulent stirring can be done with radial velocitiesabove 100 cm/sec. The oil also can be circulated in the reactor using anexternal rotary pump. The aqueous phase containing the enzyme can alsobe finely dispersed by means of ultrasound action. A dispersionapparatus can be used.

The enzymatic reaction probably takes place at the border surfacebetween the oil phase and the aqueous phase. It is the goal of all thesemeasures for mixing to create the greatest possible surface for theaqueous phase which contains the enzyme. The addition of surfactantsincreases the microdispersion of the aqueous phase. In some cases,therefore, surfactants with HLB values above 9, such as Na-dodecylsulfate, are added to the enzyme solution, as described, e.g., in EP-A 0513 709. A similar effective method for improving emulsification is theaddition of lysolecithin. The amounts added can lie in the range of0.001% to 1%, with reference to the oil. The temperature during enzymetreatment is not critical. Temperatures between 20° C. and 80° C. can beused, but the latter can only be applied for a short time. In thisaspect, a phospholipase of the invention having a good temperatureand/or low pH tolerance is used. Application temperatures of between 30°C. and 50° C. are optimal. The treatment period depends on thetemperature and can be kept shorter with an increasing temperature.Times of 0.1 to 10 hours, or, 1 to 5 hours are generally sufficient. Thereaction takes place in a degumming reactor, which can be divided intostages, as described, e.g., in DE-A 43 39 556. Therefore continuousoperation is possible, along with batch operation. The reaction can becarried out in different temperature stages. For example, incubation cantake place for 3 hours at 40° C., then for 1 hour at 60° C. If thereaction proceeds in stages, this also opens up the possibility ofadjusting different pH values in the individual stages. For example, inthe first stage the pH of the solution can be adjusted to 7, forexample, and in a second stage to 2.5, by adding citric acid. In atleast one stage, however, the pH of the enzyme solution must be below 4,or, below 3. If the pH was subsequently adjusted below this level, adeterioration of effect may be found. Therefore the citric acid can beadded to the enzyme solution before the latter is mixed into the oil.

After completion of the enzyme treatment, the enzyme solution, togetherwith the decomposition products of the NHP contained in it, can beseparated from the oil phase, in batches or continuously, e.g., by meansof centrifugation. Since the enzymes are characterized by a high levelof stability and the amount of the decomposition products contained inthe solution is slight (they may precipitate as sludge) the same aqueousenzyme phase can be used several times. There is also the possibility offreeing the enzyme of the sludge, see, e.g., DE-A 43 39 556, so that anenzyme solution which is essentially free of sludge can be used again.In one aspect of this degumming process, oils which contain less than 15ppm phosphorus are obtained. One goal is phosphorus contents of lessthan 10 ppm; or, less than 5 ppm. With phosphorus contents below 10 ppm,further processing of the oil according to the process of distillativede-acidification is easily possible. A number of other ions, such asmagnesium, calcium, zinc, as well as iron, can be removed from the oil,e.g., below 0.1 ppm. Thus, this product possesses ideal prerequisitesfor good oxidation resistance during further processing and storage.

The phospholipases and methods of the invention also can also be usedfor reducing the amount of phosphorus-containing components in vegetableand animal oils as described, e.g., in EP patent EP 0513709. In thismethod, the content of phosphorus-containing components, especiallyphosphatides, such as lecithin, and the iron content in vegetable andanimal oils, which have previously been deslimed, e.g. soya oil, arereduced by enzymatic breakdown using a phospholipase A1, A2 or B of theinvention.

The phospholipases and methods of the invention can also be used forrefining fat or oils as described, e.g., in JP 06306386. The inventionprovides processes for refining a fat or oil comprising a step ofconverting a phospholipid in a fat or an oil into a water-solublephosphoric-group-containing substance and removing this substance. Theaction of an enzyme of the invention (e.g., a PI-PLC) is utilized toconvert the phospholipid into the substance. Thus, it is possible torefine a fat or oil without carrying out an alkali refining step fromwhich industrial wastes containing alkaline waste water and a largeamount of oil are produced. Improvement of yields can be accomplishedbecause the loss of neutral fat or oil from escape with the wastes canbe reduced to zero. In one aspect, gummy substances are converted intowater-soluble substances and removed as water-soluble substances byadding an enzyme of the invention having a phospholipase C activity inthe stage of degumming the crude oil and conducting enzymatic treatment.In one aspect, the phospholipase C of the invention has an activity thatcuts ester bonds of glycerin and phosphoric acid in phospholipids. Ifnecessary, the method can comprise washing the enzyme-treated oil withwater or an acidic aqueous solution. In one aspect, the enzyme of theinvention is added to and reacted with the crude oil. The amount ofphospholipase C employed can be 10 to 10,000 units, or, about 100 to2,000 units, per 1 kg of crude oil.

The phospholipases and methods of the invention can also be used forwater-degumming processes as described, e.g., in Dijkstra, Albert J., etal., Oleagineux, Corps Gras, Lipides (1998), 5(5), 367-370. In thisexemplary method, the water-degumming process is used for the productionof lecithin and for dry degumming processes using a degumming acid andbleaching earth. This method may be economically feasible only for oilswith a low phosphatide content, e.g., palm oil, lauric oils, etc. Forseed oils having a high NHP-content, the acid refining process is used,whereby this process is carried out at the oil mill to allow gumdisposal via the meal. In one aspect, this acid refined oil is apossible “polishing” operation to be carried out prior to physicalrefining.

The phospholipases and methods of the invention can also be used fordegumming processes as described, e.g., in Dijkstra, et al., Res. Dev.Dep., N. V. Vandemoortele Coord. Cent., Izegem, Belg. JAOCS, J. Am. OilChem. Soc. (1989), 66:1002-1009. In this exemplary method, the totaldegumming process involves dispersing an acid such as H₃PO₄ or citricacid into soybean oil, allowing a contact time, and then mixing a basesuch as caustic soda or Na silicate into the acid-in-oil emulsion. Thiskeeps the degree of neutralization low enough to avoid forming soaps,because that would lead to increased oil loss. Subsequently, the oilpassed to a centrifugal separator where most of the gums are removedfrom the oil stream to yield a gum phase with minimal oil content. Theoil stream is then passed to a second centrifugal separator to removeall remaining gums to yield a dilute gum phase, which is recycled.Washing and drying or in-line alkali refining complete the process.After the adoption of the total degumming process, in comparison withthe classical alkali refining process, an overall yield improvement ofabout 0.5% is realized. The totally degummed oil can be subsequentlyalkali refined, bleached and deodorized, or bleached and physicallyrefined.

The phospholipases and methods of the invention can also be used for theremoval of nonhydratable phospholipids from a plant oil, e.g., soybeanoil, as described, e.g., in Hvolby, et al., Sojakagefabr., Copenhagen,Den., J. Amer. Oil Chem. Soc. (1971) 48:503-509. In this exemplarymethod, water-degummed oil is mixed at different fixed pH values withbuffer solutions with and without Ca⁺⁺, Mg/Ca-binding reagents, andsurfactants. The nonhydratable phospholipids can be removed in anonconverted state as a component of micelles or of mixed emulsifiers.Furthermore, the nonhydratable phospholipids are removable by conversioninto dissociated forms, e.g., by removal of Mg and Ca from thephosphatidates, which can be accomplished by acidulation or by treatmentwith Mg/Ca-complexing or Mg/Ca-precipitating reagents. Removal orchemical conversion of the nonhydratable phospholipids can result inreduced emulsion formation and in improved separation of the deacidifiedoil from the emulsion layer and the soapstock.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., Buchold, et al.,Frankfurt/Main, Germany. Fett Wissenschaft Technologie (1993), 95(8),300-304. In this exemplary process of the invention for the degumming ofedible vegetable oils, aqueous suspensions of an enzyme of theinvention, e.g., phospholipase A2, is used to hydrolyze the fatty acidbound at the sn2 position of the phospholipid, resulting in1-acyl-lysophospholipids which are insoluble in oil and thus moreamenable to physical separation. Even the addition of small amountscorresponding to about 700 lecitase units/kg oil results in a residual Pconcentration of less than 10 ppm, so that chemical refining isreplaceable by physical refining, eliminating the necessity forneutralization, soapstock splitting, and wastewater treatment.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by EnzyMax, Dahlke,Klaus, Dept. G-PDO, Lurgi Ol-Gas, Chemie, GmbH, Frankfurt, Germany.Oleagineux, Corps Gras, Lipides (1997), 4(1), 55-57. This exemplaryprocess is a degumming process for the physical refining of almost anykind of oil. By an enzymatic-catalyzed hydrolysis, phosphatides areconverted to water-soluble lysophosphatides which are separated from theoil by centrifugation. The residual phosphorus content in theenzymatically degummed oil can be as low as 2 ppm P.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Cleenewerck, et al.,N.V. Vamo Mills, Izegem, Belg. Fett Wissenschaft Technologie (1992),94:317-22; and, Clausen, Kim; Nielsen, Munk. Novozymes A/S, Den. DanskKemi (2002) 83(2):24-27. The phospholipases and methods of the inventioncan incorporate the pre-refining of vegetable oils with acids asdescribed, e.g., by Nilsson-Johansson, et al., Fats Oils Div.,Alfa-Laval Food Eng. AB, Tumba, Swed. Fett Wissenschaft Technologie(1988), 90(11), 447-51; and, Munch, Ernst W. Cereol Deutschland GmbH,Mannheim, Germany. Editor(s): Wilson, Richard F. Proceedings of theWorld Conference on Oilseed Processing Utilization, Cancun, MX, Nov.12-17, (2001), Meeting Date 2000, 17-20.

The phospholipases and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Jerzewska, et al.,Inst. Przemyslu Miesnego i Tluszczowego, Warsaw, Pol., Tluszcze Jadalne(2001), 36(3/4), 97-110. In this process of the invention, enzymaticdegumming of hydrated low-erucic acid rapeseed oil is by use of aphospholipase A2 of the invention. The enzyme can catalyze thehydrolysis of fatty acid ester linkages to the central carbon atom ofthe glycerol moiety in phospholipids. It can hydrolyze non-hydratablephospholipids to their corresponding hydratable lyso-compounds. With anonpurified enzyme preparation, better results can be achieved with theaddition of 2% preparation for 4 hours (87% P removal).

In another exemplary process of the invention for oil degumming (or anoil degumming process using an enzyme of the invention), an acidicpolymer, e.g., an alginate or pectin, is added. In this oil degummingprocess of the invention, an acidic polymer (e.g. alginic acid or pectinor a more soluble salt form) is added to the crude oil with a low amountof water (e.g., in a range of between about 0.5 to 5%). In this aspect,the acidic polymers can reduce and/or disrupt phospholipid-metalcomplexes by binding calcium and/or magnesium in the crude oil, therebyimproving the solubility of nonhydratable phospholipids. In alternativeaspects, these phospholipids will move to the oil/water interface orenter the aqueous phase and either be converted to diacylglycerol andthe corresponding side chain or the intact phospholipid will be removedby subsequent centrifugation as a component of the heavy phase. Thepresence of the acidic polymer in the aqueous phase can also increasethe density of the aqueous phase and result in an improved separation ofthe heavy phase from the oil (light) phase.

One exemplary process of the invention for oil degumming (or an oildegumming process using an enzyme of the invention) alters thedeodorization procedure to get a diacylglycerol (DAG) fraction. Inalternative aspect, if necessary or desired, following enzyme-assisteddegumming, the deodorization conditions (temperature, pressure,configuration of the distillation apparatus) can be modified with thegoal of improving the separation of the free fatty acids (FFA) from thediacylglycerol/triacylglycerol fraction or further modified to separatethe diacylglycerol from the triacylglycerol fraction. As a result ofthese modifications, using this method of the invention, it is possibleto obtain food grade FFA and diacylglycerol if an enzyme of theinvention (e.g., a phosphatase, or, a PLC or a combination of PLC andphosphatases) are used to degum edible oil in a physical refiningprocess.

In various aspects, practicing the methods of the invention as describedherein (or using the enzymes of the invention), have advantages such as:decrease or eliminate solvent and solvent recovery; lower capital costs;decrease downstream refining costs, decrease chemical usage, equipment,process time, energy (heat) and water usage/wastewater generation;produce higher quality oil; expeller pressed oil may be used withoutrefining in some cooking and sautéing applications (this pressed oil mayhave superior stability, color and odor characteristics and hightocopherol content); produce higher quality meal; produce a lower fatcontent in meal (currently, meal coming out of mechanical press causesdigestion problems in ruminants); produce improved nutritionalattributes—reduced levels of glucosinolates, tannins, sinapine, phyticacid (as described, e.g., in Technology and Solvents for ExtractingOilseeds and Nonpetroleum Oils, AOCS 1997).

In one aspect, the invention provides methods for refining vegetableoils (e.g., soybean oil, corn oil, cottonseed oil, palm oil, peanut oil,rapeseed oil, safflower oil, sunflower seed oil, sesame seed oil, ricebran oil, coconut oil or canola oil) and their byproducts, and processesfor deodorizing lecithin, for example, as described in U.S. Pat. No.6,172,248, or 6,172,247, wherein the methods comprise use of at leastone enzyme of the invention, e.g., a phospholipase C of the invention.Thus, the invention provides lecithin and vegetable oils comprising atleast one enzyme of the invention. In an exemplary organic acid refiningprocess, vegetable oil is combined with a dilute aqueous organic acidsolution and subjected to high shear to finely disperse the acidsolution in the oil. The resulting acid-and-oil mixture is mixed at lowshear for a time sufficient to sequester contaminants into a hydratedimpurities phase, producing a purified vegetable oil phase. In thisexemplary process, a mixer or recycle system (e.g., recycle water tank)and/or a phosphatide or lecithin storage tank can be used, e.g., asdescribed in U.S. Pat. No. 4,240,972, 4,049,686, 6,172,247 or 6,172,248.These processes can be conducted as a batch or continuous process. Crudeor degummed vegetable oil can be supplied from a storage tank (e.g.,through a pump) and can be heated. The vegetable oil to be purified canbe either crude or “degummed” oil.

In one aspect, phosphatidylinositol-PLC (PI-PLC) enzymes of theinvention are used for vegetable oil degumming PI-PLC enzymes of theinvention can be used alone or in combination with other enzymes (forinstance PLC, PLD, phosphatase enzymes of the invention) to improve oilyield during the degumming of vegetable oils (including soybean, canola,and sunflower). The PI-PLC may preferentially convertphosphatidylinositol to 1,2-diacylglycerol (DAG) and phosphoinositol butit may also demonstrate activity on other phospholipids includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, orphosphatidic acid, or a combination thereof. The improvement in yieldwill be realized as an increase in the amount of DAG in theenzyme-treated vegetable oil and an increase in neutral oil, due to adecrease in the amount of oil entrained in the smaller gum fraction thatresults from enzyme treatment of the vegetable oil.

Enzymatic Processing of Oilseeds

The invention provides compositions (e.g., enzymes) and methods forenzymatic processing of oilseeds, including soybean, canola, coconut,avocado and olive paste. In one aspect, these processes of the inventioncan increase the oil yield and to improve the nutritional quality of theobtained meals. In some aspects, enzymatic processing of oilseeds usingthe enzymes and methods of the invention will provide economical andenvironmental benefits, as well as alternative technologies for oilextraction and processing food for human and animal consumption. Inalternative aspects, the processes of the invention comprise use ofphospholipases of the invention, other phospholipases, proteases,phosphatases, phytases, xylanases, amylases (e.g., α-amylases),glucanases (e.g., β-glucanases), polygalacturonases, galactolipases,cellulases, hemicellulases, pectinases and other plant cell walldegrading enzymes, as well as mixed enzyme preparations and celllysates.

In alternative aspects, the processes of the invention can be practicedin conjunction with other processes, e.g., enzymatic treatments, e.g.,with carbohydrases, including cellulase, hemicellulase and other sidedegrading activities, or, chemical processes, e.g., hexane extraction ofsoybean oil. The enzymatic treatment can increase the oil extractabilityby 8-10% when the enzymatic treatment is carried out prior to thesolvent extraction.

In alternative aspects, the processes of the invention can be practicedwith aqueous extraction processes. The aqueous extraction methods can beenvironmentally cleaner alternative technologies for oil extraction. Lowextraction yields of aqueous process can be overcome by using enzymesthat hydrolyze the structural polysaccharides forming the cell wall ofoilseeds, or that hydrolyze the proteins which form the cell and lipidbody membranes, e.g., utilizing digestions comprising cellulase,hemicellulase, and/or protopectinase for extraction of oil from soybeancells. In one aspect, methods are practiced with an enzyme of theinvention as described by Kasai (2003) J. Agric. Food Chem.51:6217-6222, who reported that the most effective enzyme to digest thecell wall was cellulase.

In one aspect, proteases are used in combination with the methods of theinvention. The combined effect of operational variables and enzymeactivity of protease and cellulase on oil and protein extraction yieldscombined with other process parameters, such as enzyme concentration,time of hydrolysis, particle size and solid-to-liquid ratio has beenevaluated. In one aspect, methods are practiced with an enzyme of theinvention as described by Rosenthal (2001) Enzyme and Microb. Tech.28:499-509, who reported that use of protease can result insignificantly higher yields of oil and protein over the control whenheat treated flour is used.

In one aspect, complete protein, pectin, and hemicellulose extractionare used in combination with the methods of the invention. The plantcell consists of a series of polysaccharides often associated with orreplaced by proteins or phenolic compounds. Most of these carbohydratesare only partially digested or poorly utilized by the digestive enzymes.The disruption of these structures through processing or degradingenzymes can improve their nutrient availability. In one aspect, methodsare practiced with an enzyme of the invention as described by Ouhida(2002) J. Agric. Food Chem. 50:1933-1938, who reported that asignificant degradation of the soybean cell wall cellulose (up to 20%)has been achieved after complete protein, pectin, and hemicelluloseextraction.

In one aspect, the methods of the invention further compriseincorporation of various enzymatic treatments in the treatment of seeds,e.g., canola seeds, these treatments comprising use of proteases,cellulases, and hemicellulases (in various combinations with each otherand with one or more enzymes of the invention). For example, the methodscan comprise enzymatic treatments of canola seeds at 20 to 40 moistureduring the incubation with enzymes prior to a conventional process; asdescribed, e.g., by Sosulski (1990) Proc. Can. Inst. Food Sci. Technol.3:656. The methods of the invention can further comprise incorporationof proteases, amylases, polygalacturonases (in various combinations witheach other and with one or more enzymes of the invention) to hydrolyzecellular material in coconut meal and release the coconut oil, which canbe recovered by centrifugation, as described, e.g., by McGlone (1986) J.of Food Sci. 51:695-697. The methods of the invention can furthercomprise incorporation of pectinases, amylases, proteases, cellulases indifferent combinations (with each other and with one or more enzymes ofthe invention) to result in significant yield improvement (˜70% in thebest case) during enzymatic extraction of avocado oil, as described,e.g., by Buenrostro (1986) Biotech. Letters 8(7):505-506. In processesof the invention for olive oil extraction, olive paste is treated withcellulase, hemicellulase, poligalacturonase, pectin-methyltransferase,protease and their combinations (with each other and with one or moreenzymes of the invention), as described, e.g., by Montedoro (1976) ActaVitamin. Enzymol. (Milano) 30:13.

Purification of Phytosterols from Vegetable Oils

The invention provides methods for purification of phytosterols andtriterpenes, or plant sterols, from vegetable oils. Phytosterols thatcan be purified using phospholipases and methods of the inventioninclude β-sitosterol, campesterol, stigmasterol, stigmastanol,β-sitostanol, sitostanol, desmosterol, chalinasterol, poriferasterol,clionasterol and brassicasterol. Plant sterols are importantagricultural products for health and nutritional industries. Thus,phospholipases and methods of the invention are used to make emulsifiersfor cosmetic manufacturers and steroidal intermediates and precursorsfor the production of hormone pharmaceuticals. Phospholipases andmethods of the invention are used to make (e.g., purify) analogs ofphytosterols and their esters for use as cholesterol-lowering agentswith cardiologic health benefits. Phospholipases and methods of theinvention are used to purify plant sterols to reduce serum cholesterollevels by inhibiting cholesterol absorption in the intestinal lumen.Phospholipases and methods of the invention are used to purify plantsterols that have immunomodulating properties at extremely lowconcentrations, including enhanced cellular response of T lymphocytesand cytotoxic ability of natural killer cells against a cancer cellline. Phospholipases and methods of the invention are used to purifyplant sterols for the treatment of pulmonary tuberculosis, rheumatoidarthritis, management of HIV-infested patients and inhibition of immunestress, e.g., in marathon runners.

Phospholipases and methods of the invention are used to purify sterolcomponents present in the sterol fractions of commodity vegetable oils(e.g., coconut, canola, cocoa butter, corn, cottonseed, linseed, olive,palm, peanut, rice bran, safflower, sesame, soybean, sunflower oils),such as sitosterol (40.2-92.3%), campesterol (2.6-38.6%), stigmasterol(0-31%) and 5-avenasterol (1.5-29%).

Methods of the invention can incorporate isolation of plant-derivedsterols in oil seeds by solvent extraction with chloroform-methanol,hexane, methylene chloride, or acetone, followed by saponification andchromatographic purification for obtaining enriched total sterols.Alternatively, the plant samples can be extracted by supercritical fluidextraction with supercritical carbon dioxide to obtain total lipidextracts from which sterols can be enriched and isolated. For subsequentcharacterization and quantification of sterol compounds, the crudeisolate can be purified and separated by a wide variety ofchromatographic techniques including column chromatography (CC), gaschromatography, thin-layer chromatography (TLC), normal phasehigh-performance liquid chromatography (HPLC), reversed-phase HPLC andcapillary electro-chromatography. Of all chromatographic isolation andseparation techniques, CC and TLC procedures employ the most accessible,affordable and suitable for sample clean up, purification, qualitativeassays and preliminary estimates of the sterols in test samples.

Phytosterols are lost in the vegetable oils lost as byproducts duringedible oil refining processes. Phospholipases and methods of theinvention use phytosterols isolated from such byproducts to makephytosterol-enriched products isolated from such byproducts. Phytosterolisolation and purification methods of the invention can incorporate oilprocessing industry byproducts and can comprise operations such asmolecular distillation, liquid-liquid extraction and crystallization.

Methods of the invention can incorporate processes for the extraction oflipids to extract phytosterols. For example, methods of the inventioncan use nonpolar solvents as hexane (commonly used to extract most typesof vegetable oils) quantitatively to extract free phytosterols andphytosteryl fatty-acid esters. Steryl glycosides and fatty-acylatedsteryl glycosides are only partially extracted with hexane, andincreasing polarity of the solvent gave higher percentage of extraction.One procedure that can be used is the Bligh and Dyer chloroform-methanolmethod for extraction of all sterol lipid classes, includingphospholipids. One exemplary method to both qualitatively separate andquantitatively analyze phytosterol lipid classes comprises injection ofthe lipid extract into HPLC system.

Phospholipases and methods of the invention can be used to removesterols from fats and oils, as described, e.g., in U.S. Pat. No.6,303,803. This is a method for reducing sterol content ofsterol-containing fats and oils. It is an efficient and cost effectiveprocess based on the affinity of cholesterol and other sterols foramphipathic molecules that form hydrophobic, fluid bilayers, such asphospholipid bilayers. Aggregates of phospholipids are contacted with,for example, a sterol-containing fat or oil in an aqueous environmentand then mixed. The molecular structure of this aggregated phospholipidmixture has a high affinity for cholesterol and other sterols, and canselectively remove such molecules from fats and oils. The aqueousseparation mixture is mixed for a time sufficient to selectively reducethe sterol content of the fat/oil product through partitioning of thesterol into the portion of phospholipid aggregates. The sterol-reducedfat or oil is separated from the aqueous separation mixture.Alternatively, the correspondingly sterol-enriched fraction also may beisolated from the aqueous separation mixture. These steps can beperformed at ambient temperatures, costs involved in heating areminimized, as is the possibility of thermal degradation of the product.Additionally, a minimal amount of equipment is required, and since allrequired materials are food grade, the methods require no specialprecautions regarding handling, waste disposal, or contamination of thefinal product(s).

Phospholipases and methods of the invention can be used to removesterols from fats and oils, as described, e.g., in U.S. Pat. No.5,880,300. Phospholipid aggregates are contacted with, for example, asterol-containing fat or oil in an aqueous environment and then mixed.Following adequate mixing, the sterol-reduced fat or oil is separatedfrom the aqueous separation mixture. Alternatively, the correspondinglysterol-enriched phospholipid also may be isolated from the aqueousseparation mixture. Plant (e.g., vegetable) oils contain plant sterols(phytosterols) that also may be removed using the methods of the presentinvention. This method is applicable to a fat/oil product at any stageof a commercial processing cycle. For example, the process of theinvention may be applied to refined, bleached and deodorized oils (“RBDoils”), or to any stage of processing prior to attainment of RBD status.Although RBD oil may have an altered density compared to pre-RBD oil,the processes of the invention are readily adapted to either RBD orpre-RBD oils, or to various other fat/oil products, by variation ofphospholipid content, phospholipid composition, phospholipid:waterratios, temperature, pressure, mixing conditions, and separationconditions as described below.

Alternatively, the enzymes and methods of the invention can be used toisolate phytosterols or other sterols at intermediate steps in oilprocessing. For example, it is known that phytosterols are lost duringdeodorization of plant oils. A sterol-containing distillate fractionfrom, for example, an intermediate stage of processing can be subjectedto the sterol-extraction procedures described above. This provides asterol-enriched lecithin or other phospholipid material that can befurther processed in order to recover the extracted sterols.

Detergent Compositions

The invention provides detergent compositions comprising one or morephospholipase of the invention, and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;6,365,561; 6,380,147. The detergent compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The invention also provides methodscapable of a rapid removal of gross food soils, films of food residueand other minor food compositions using these detergent compositions.Phospholipases of the invention can facilitate the removal of stains bymeans of catalytic hydrolysis of phospholipids. Phospholipases of theinvention can be used in dishwashing detergents in textile launderingdetergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof phospholipase present in the final solution ranges from about 0.001mg to 0.5 mg per gram of the detergent composition. The particularenzyme chosen for use in the process and products of this inventiondepends upon the conditions of final utility, including the physicalproduct form, use pH, use temperature, and soil types to be degraded oraltered. The enzyme can be chosen to provide optimum activity andstability for any given set of utility conditions. In one aspect, thepolypeptides of the present invention are active in the pH ranges offrom about 4 to about 12 and in the temperature range of from about 20°C. to about 95° C. The detergents of the invention can comprisecationic, semi-polar nonionic or zwitterionic surfactants; or, mixturesthereof.

Phospholipases of the present invention can be formulated into powderedand liquid detergents having pH between 4.0 and 12.0 at levels of about0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as known proteases,cellulases, lipases or endoglycosidases, as well as builders andstabilizers. The addition of phospholipases of the invention toconventional cleaning compositions does not create any special uselimitation. In other words, any temperature and pH suitable for thedetergent is also suitable for the present compositions as long as thepH is within the above range, and the temperature is below the describedenzyme's denaturing temperature. In addition, the polypeptides of theinvention can be used in a cleaning composition without detergents,again either alone or in combination with builders and stabilizers.

The present invention provides cleaning or disinfecting compositionsincluding detergent and/or disinfecting compositions for cleaning and/ordisinfecting hard surfaces, detergent compositions for cleaning and/ordisinfecting fabrics, dishwashing compositions, oral cleaningcompositions, denture cleaning compositions, and/or contact lenscleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a phospholipase of the inventionunder conditions sufficient for washing. A phospholipase of theinvention may be included as a detergent additive. The detergentcomposition of the invention may, for example, be formulated as a handor machine laundry detergent composition comprising a phospholipase ofthe invention. A laundry additive suitable for pre-treatment of stainedfabrics can comprise a phospholipase of the invention. A fabric softenercomposition can comprise a phospholipase of the invention.Alternatively, a phospholipase of the invention can be formulated as adetergent composition for use in general household hard surface cleaningoperations. In alternative aspects, detergent additives and detergentcompositions of the invention may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, another phospholipase, acarbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, agalactanase, a xylanase, an oxidase, e.g., a lactase, and/or aperoxidase. The properties of the enzyme(s) of the invention are chosento be compatible with the selected detergent (i.e. pH-optimum,compatibility with other enzymatic and non-enzymatic ingredients, etc.)and the enzyme(s) is present in effective amounts. In one aspect,phospholipase enzymes of the invention are used to remove malodorousmaterials from fabrics. Various detergent compositions and methods formaking them that can be used in practicing the invention are describedin, e.g., U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038;6,309,871; 6,204,232; 6,197,070; 5,856,164.

Waste Treatment

The phospholipases of the invention can be used in waste treatment. Inone aspect, the invention provides a solid waste digestion process usingphospholipases of the invention. The methods can comprise reducing themass and volume of substantially untreated solid waste. Solid waste canbe treated with an enzymatic digestive process in the presence of anenzymatic solution (including phospholipases of the invention) at acontrolled temperature. The solid waste can be converted into aliquefied waste and any residual solid waste. The resulting liquefiedwaste can be separated from said any residual solidified waste. Seee.g., U.S. Pat. No. 5,709,796.

Detoxification

The phospholipases (e.g., PI-PLCs of the invention) can be used indetoxification processes, e.g., for the detoxification of endotoxins,e.g., compositions comprising lipopolysaccharides (LPS), and, theinvention provides detoxification processes using at least one enzyme ofthe invention, e.g., a polypeptide comprising a sequence as set forth inSEQ ID NO:6 and having one or more mutations as set forth in Tables 12to 15, or an enzymatically active fragment thereof.

In one aspect, a phospholipase of the invention is used to detoxify alipopolysaccharide (LPS). In one aspect, this detoxification is bydeacylation of 2′ and/or 3′ fatty acid chains from lipid A. In oneaspect, a phospholipase (e.g., a PI-PLC) of the invention is used tohydrolyze a 2′-lauroyl and/or a 3′-myristoyl chain from a lipid, e.g., alipid A (e.g., from a bacterial endotoxin). In one aspect, the processof the invention is used to destroy an endotoxin, e.g., a toxin from agram negative bacteria, as from E. coli. In one aspect, a phospholipase(e.g., a PI-PLC) of the invention is used to ameliorate the effects oftoxin poisoning (e.g., from an on-going gram negative infection), or, toprophylactically to prevent the effects of endotoxin during an infection(e.g., an infection in an animal or a human). Accordingly, the inventionprovides a pharmaceutical composition comprising a phospholipase (e.g.,PI-PLC) of the invention, and method using a hydrolase of the invention,for the amelioration or prevention of lipopolysaccharide (LPS) toxiceffects, e.g., during sepsis.

Processing Foods

The phospholipases of the invention can be used to process foods, e.g.,to change their stability, shelf-life, flavor, texture, improve on theirnutritional status, and the like. For example, in one aspect,phospholipases of the invention are used to generate acidicphospholipids for controlling bitter taste in foods.

In one aspect, the invention provides cheese-making processes usingphospholipases of the invention (and, thus, the invention also providescheeses comprising phospholipases of the invention). In one aspect, theenzymes of the invention (e.g., phospholipase A, lysophospholipase or acombination thereof) are used to process cheeses for flavor enhancement,to increase yield and/or for “stabilizing” cheeses, e.g., by reducingthe tendency for “oil-off,” or, in one aspect, the enzymes of theinvention are used to produce cheese from cheese milk. These processesof the invention can incorporate any method or protocol, e.g., asdescribed, e.g., in U.S. Pat. Nos. 6,551,635, and 6,399,121, WO03/070013, WO 00/054601. For example, in one aspect, the phospholipasesof the invention are used to stabilize fat emulsion in milk ormilk-comprising compositions, e.g. cream, and are used to stabilize milkcompositions, e.g. for the manufacturing of creams or cream liquors. Inone aspect, the invention provides a process for enhancing the favor ofa cheese using at least one enzyme of the invention, the processcomprising incubating a protein, a fat and a protease and a lipase in anaqueous medium under conditions that produce an enhanced cheese flavor(e.g., reduced bitterness), e.g., as described in WO 99/66805. In oneaspect, phospholipases of the invention are used to enhance flavor in acheese (e.g., a curd) by mixing with water, a protease, and a lipase (ofthe invention) at an elevated temperature, e.g., between about 75° C. to95° C., as described, e.g., in U.S. Pat. No. 4,752,483. In one aspect,phospholipases of the invention are used to accelerate cheese aging byadding an enzyme of the invention (e.g., a lipase or a phospholipase) toa cheese (e.g., a cheese milk) before adding a coagulant to the milk,or, adding an enzyme of the invention to a curd with salt beforepressing, e.g., as described, e.g., in U.S. Pat. No. 4,707,364. In oneaspect, a lipase of the invention is used degrade a triglyceride in milkfat to liberate free fatty acids, resulting in flavor enhancement. Aprotease also can be used in any of these processes of the invention,see, e.g., Brindisi (2001) J. of Food Sci. 66:1100-1107. In anotheraspect, a combination of esterases, lipases, phospholipases and/orproteases can be used in these or any process of the invention.

In one aspect, a phospholipase of the invention is used to reduce thecontent of phosphorus components in a food, e.g., an oil, such as avegetable oil having a high non-hydratable phosphorus content, e.g., asdescribed in WO 98/26057.

Biomass Conversion and Production of Clean Biofuels

The invention provides polypeptides, including enzymes (phospholipases(PLs), e.g., PLAs, PLCs or PLDs of the invention) and antibodies of theinvention, and methods for the conversion of a biomass or anylignocellulosic material (e.g., any composition comprising cellulose,hemicellulose and lignin), to a fuel (e.g., bioethanol, biopropanol,biobutanol, biopropanol, biomethanol, biodiesel), in addition to feeds,foods and chemicals. For example, in alternative embodiment, enzyme(s)of the invention used for biomass conversion and for the production ofbiofuels can have one or more phospholipase activities, including aphospholipase C (PLC) activity; a PI-PLC activity, a phospholipase A(PLA) activity, such as a phospholipase A1 or phospholipase A2 activity;a phospholipase D (PLD) activity, such as a phospholipase D1 or aphospholipase D2 activity; a phospholipase B (PLB) activity, e.g., aphospholipase and a lysophospholipase (LPL) activity or a phospholipaseand a lysophospholipase-transacylase (LPTA) activity or a phospholipaseand a lysophospholipase (LPL) activity andlysophospholipase-transacylase (LPTA) activity; or patatin activity, ora combination thereof.

Thus, the compositions and methods of the invention provide effectiveand sustainable alternatives or adjuncts to use of petroleum-basedproducts, e.g., as a mixture of a biofuel such as biomethanol,bioethanol, biopropanol, biobutanol, and the like, to diesel fuel,gasoline, kerosene and the like. The invention provides organismsexpressing enzymes of the invention for participation in chemical cyclesinvolving natural biomass conversion. In one aspect, enzymes and methodsfor the conversion are used in enzyme ensembles for phospholipidprocessing. The invention provides methods for discovering andimplementing the most effective of enzymes to enable these important new“biomass conversion” and alternative energy industrial processes.

The compositions and methods of the invention can be used to provideeffective and sustainable alternatives or adjuncts to use ofpetroleum-based products, e.g., as a mixture of bioethanol, biopropanol,biobutanol, biopropanol, biomethanol and/or biodiesel and gasoline. Theinvention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.The invention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

The invention provides methods, enzymes and mixtures of enzymes or“cocktails” of the invention, for processing a material, e.g. a biomassmaterial, comprising a cellooligsaccharide, an arabinoxylan oligomer, alignin, a lignocellulose, a xylan, a glucan, a cellulose and/or afermentable sugar comprising contacting the composition with apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention, wherein optionally the material is derived from anagricultural crop (e.g., wheat, barley, potatoes, switchgrass, poplarwood), is a byproduct of a food or a feed production, is alignocellulosic waste product, or is a plant residue or a waste paper orwaste paper product, and optionally the plant residue comprise stems,leaves, hulls, husks, corn or corn cobs, corn stover, corn fiber, hay,straw (e.g. rice straw or wheat straw), sugarcane bagasse, sugar beetpulp, citrus pulp, and citrus peels, wood, wood thinnings, wood chips,wood pulp, pulp waste, wood waste, wood shavings and sawdust,construction and/or demolition wastes and debris (e.g. wood, woodshavings and sawdust), and optionally the paper waste comprisesdiscarded or used photocopy paper, computer printer paper, notebookpaper, notepad paper, typewriter paper, newspapers, magazines, cardboardand paper-based packaging materials, and recycled paper materials. Inaddition, urban wastes, e.g. the paper fraction of municipal solidwaste, municipal wood waste, and municipal green waste, along with othermaterials containing sugar, starch, and/or cellulose can be used. Inalternative aspects, the processing of the material, e.g. the biomassmaterial, generates a bioalcohol, e.g., a bioethanol, biomethanol,biobutanol or biopropanol.

Alternatively, the polypeptide of the invention may be expressed in thebiomass plant material or feedstock itself.

The methods of the invention also include taking a processed, or“converted” (e.g., by process comprising use of an enzyme of thisinvention) biomass or plant material, e.g., a lipid-comprising or alignocellulosic material (processed by, e.g., enzymes of the invention)and making it into a fuel (e.g. a bioalcohol, e.g., a bioethanol,biomethanol, biobutanol or biopropanol, or biodiesel) by fermentation(e.g., by yeast) and/or by chemical synthesis. In one aspect, theproduced sugars are fermented and/or the non-fermentable products aregasified.

The methods of the invention also include converting algae, vegetableoil such as virgin vegetable oils or waste vegetable oils, animal fatsand greases (e.g. tallow, lard, and yellow grease), or sewage, usingenzymes of the invention, and making it into a fuel (e.g. a bioalcohol,e.g., a bioethanol, biomethanol, biobutanol or biopropanol, orbiodiesel) by fermentation and/or by chemical synthesis or conversion.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, making and in someaspects secreting recombinant enzymes of the invention) can be used inor included/integrated at any stage of any biomass conversion process,e.g., at any one step, several steps, or included in all of the steps,or all of the following methods of biomass conversion processes, or allof these biofuel alternatives:

-   -   Direct combustion: the burning of material by direct heat and is        the simplest biomass technology; can be very economical if a        biomass source is nearby.    -   Pyrolysis: is the thermal degradation of biomass by heat in the        absence of oxygen. In one aspect, biomass is heated to a        temperature between about 800 and 1400 degrees Fahrenheit, but        no oxygen is introduced to support combustion resulting in the        creation of gas, fuel oil and charcoal.    -   Gasification: biomass can be used to produce methane through        heating or anaerobic digestion. Syngas, a mixture of carbon        monoxide and hydrogen, can be derived from biomass.    -   Landfill Gas: is generated by the decay (anaerobic digestion) of        buried garbage in landfills. When the organic waste decomposes,        it generates gas consisting of approximately 50% methane, the        major component of natural gas.    -   Anaerobic digestion: converts organic matter to a mixture of        methane, the major component of natural gas, and carbon dioxide.        In one aspect, biomass such as waterwaste (sewage), manure, or        food processing waste, is mixed with water and fed into a        digester tank without air.    -   Fermentation        -   Alcohol Fermentation: fuel alcohol is produced by converting            cellulosic mass and/or starch to sugar, fermenting the sugar            to alcohol, then separating the alcohol water mixture by            distillation. Feedstocks such as dedicated crops (e.g.,            wheat, barley, potatoes, switchgrass, poplar wood),            agricultural residues and wastes (e.g. rice straw, corn            stover, wheat straw, sugarcane bagasse, rice hulls, corn            fiber, sugar beet pulp, citrus pulp, and citrus peels),            forestry wastes (e.g. hardwood and softwood thinnings,            hardwood and softwood residues from timber operations, wood            shavings, and sawdust), urban wastes (e.g. paper fraction of            municipal solid waste, municipal wood waste, municipal green            waste), wood wastes (e.g. saw mill waste, pulp mill waste,            construction waste, demolition waste, wood shavings, and            sawdust), and waste paper or other materials containing            sugar, starch, and/or cellulose can be converted to sugars            and then to alcohol by fermentation with yeast.            Alternatively, materials containing sugars can be converted            directly to alcohol by fermentation.    -   Transesterification: An exemplary reaction for converting oil to        biodiesel is called transesterification. The transesterification        process reacts an alcohol (like methanol) with the triglyceride        oils contained in vegetable oils, animal fats, or recycled        greases, forming fatty acid alkyl esters (biodiesel) and        glycerin. The reaction requires heat and a strong base catalyst,        such as sodium hydroxide or potassium hydroxide.    -   Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters        made from vegetable oils, animal fats or recycled greases.        Biodiesel can be used as a fuel for vehicles in its pure form,        but it is usually used as a petroleum diesel additive to reduce        levels of particulates, carbon monoxide, hydrocarbons and air        toxics from diesel-powered vehicles.    -   Hydrolysis: includes hydrolysis of a compound, e.g., a biomass,        such as a lignocellulosic material, catalyzed using an enzyme of        the instant invention.    -   Congeneration: is the simultaneous production of more than one        form of energy using a single fuel and facility. In one aspect,        biomass cogeneration has more potential growth than biomass        generation alone because cogeneration produces both heat and        electricity.

In one aspect, the polypeptides of the invention have hydrolaseactivity, e.g., phospholipase, patatin and/or other related enzymaticactivity for generating a fuel (e.g. a bioalcohol, e.g., a bioethanol,biomethanol, biobutanol or biopropanol, or biodiesel) from an organicmaterial, e.g., a biomass, such as compositions derived from plants andanimals, including any agricultural crop or other renewable feedstock,an agricultural residue or an animal waste, the organic components ofmunicipal and industrial wastes, or construction or demolition wastes ordebris, or microorganisms such as algae or yeast.

In one aspect, polypeptides of the invention are used in processes forconverting any biomass, e.g., an animal, algae and/or plant biomassincluding lipid-comprising or lignocellulosic biomass to a fuel (e.g. abioalcohol, e.g., a bioethanol, biomethanol, biobutanol or biopropanol,or biodiesel), or otherwise are used in processes for hydrolyzing ordigesting biomaterials such that they can be used as a fuel (e.g. abioalcohol, e.g., a bioethanol, biomethanol, biobutanol or biopropanol,or biodiesel), or for making it easier for the biomass to be processedinto a fuel.

Enzymes of the invention, including the mixture of enzymes or“cocktails” of the invention, can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In a large biodiesel plants of the invention, the glycerincan be further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Fuels (including bioalcohols such as bioethanols, biomethanols,biobutanols or biopropanols, or biodiesels) made using the polypeptidesof the invention, including the mixture of enzymes or “cocktails” of theinvention, can be used with fuel oxygenates to improve combustioncharacteristics. Adding oxygen results in more complete combustion,which reduces carbon monoxide emissions. This is another environmentalbenefit of replacing petroleum fuels with biofuels (e.g., a fuel of theinvention). A biofuel made using the compositions and/or methods of thisinvention can be blended with gasoline to form an E10 blend (about 5% to10% ethanol and about 90% to 95% gasoline), but it can be used in higherconcentrations such as E85 or in its pure form. A biofuel made using thecompositions and/or methods of this invention can be blended withpetroleum diesel to form a B20 blend (20% biodiesel and 80% petroleumdiesel), although other blend levels can be used up to B100 (purebiodiesel).

The invention also provides processes for making biofuels (includingbioalcohols such as bioethanols, biomethanols, biobutanols orbiopropanols, or biodiesels) from compositions comprising any biomass,e.g., an animal, algae and/or plant biomass including lipid-comprisingor lignocellulosic biomass. The biomass material can be obtained fromagricultural crops, as a byproduct of food or feed production, or aslignocellulosic waste products, such as plant residues, waste paper orconstruction and/or demolition wastes or debris. Examples of suitableplant sources or plant residues for treatment with polypeptides of theinvention include kelp, algae, grains, seeds, stems, leaves, hulls,husks, corn cobs, corn stover, straw, grasses (e.g., Indian grass, suchas Sorghastrum nutans; or, switch grass, e.g., Panicum species, such asPanicum virgatum), and the like, as well as wood, wood chips, wood pulp,and sawdust. Examples of paper waste suitable for treatment withpolypeptides of the invention include discard photocopy paper, computerprinter paper, notebook paper, notepad paper, typewriter paper, and thelike, as well as newspapers, magazines, cardboard, and paper-basedpackaging materials. Examples of construction and demolition wastes anddebris include wood, wood scraps, wood shavings and sawdust.

In one embodiment, the enzymes, including the mixture of enzymes or“cocktails” of the invention, and methods of the invention can be usedin conjunction with more “traditional” means of making ethanol,methanol, propanol, butanol, propanol and/or diesel from biomass, e.g.,as methods comprising hydrolyzing lipids and/or lignocellulosicmaterials by subjecting dried any biomass, e.g., an animal, algae and/orplant biomass including lipid-comprising or lignocellulosic biomassmaterial in a reactor to a catalyst comprised of a dilute solution of astrong acid and a metal salt; this can lower the activation energy, orthe temperature, of cellulose hydrolysis to obtain higher sugar yields;see, e.g., U.S. Pat. Nos. 6,660,506 and 6,423,145.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises hydrolyzing any biomass, e.g., an animal, algaeand/or plant biomass including lipid-comprising or lignocellulosicbiomass containing hemicellulose, cellulose and lignin, or any otherpolysaccharide that can be hydrolyzed by an enzyme of this invention, bysubjecting the material to a first stage hydrolysis step in an aqueousmedium at a temperature and a pressure chosen to effect primarilydepolymerization of hemicellulose without major depolymerization ofcellulose to glucose. This step results in a slurry in which the liquidaqueous phase contains dissolved monosaccharides resulting fromdepolymerization of hemicellulose and a solid phase containing celluloseand lignin. A second stage hydrolysis step can comprise conditions suchthat at least a major portion of the cellulose is depolymerized, suchstep resulting in a liquid aqueous phase containing dissolved/solubledepolymerization products of cellulose. See, e.g., U.S. Pat. No.5,536,325. Enzymes of the invention (including the invention's mixtures,or “cocktails” of enzymes) can be added at any stage of this exemplaryprocess.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises processing a any biomass, e.g., an animal, algaeand/or plant biomass including lipid-comprising or lignocellulosicbiomass material by one or more stages of dilute acid hydrolysis withabout 0.4% to 2% strong acid; and treating an unreacted solidlignocellulosic component of the acid hydrolyzed biomass material byalkaline delignification to produce precursors for biodegradablethermoplastics and derivatives. See, e.g., U.S. Pat. No. 6,409,841.Enzymes of the invention can be added at any stage of this exemplaryprocess.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises prehydrolyzing any biomass, e.g., an animal, algaeand/or plant biomass including lipid-comprising or lignocellulosicbiomass material in a prehydrolysis reactor; adding an acidic liquid tothe solid material (e.g., lignocellulosic material) to make a mixture;heating the mixture to reaction temperature; maintaining reactiontemperature for time sufficient to fractionate the lignocellulosicmaterial into a solubilized portion containing at least about 20% of thelignin from the lignocellulosic material and a solid fraction containingcellulose; removing a solubilized portion from the solid fraction whileat or near reaction temperature wherein the cellulose in the solidfraction is rendered more amenable to enzymatic digestion; andrecovering a solubilized portion. See, e.g., U.S. Pat. No. 5,705,369.Enzymes of the invention can be added at any stage of this exemplaryprocess.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one aspect, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol blends.In one aspect, a co-solvent is biomass-derived 2-methyltetrahydrofuran(MTHF). See, e.g., U.S. Pat. No. 6,712,866.

In one aspect, methods of the invention for the enzymatic degradation ofany biomass, e.g., an animal, algae and/or plant biomass includinglipid-comprising or lignocellulosic biomass, e.g., for production ofbiofuels (including bioalcohols such as bioethanols, biomethanols,biobutanols or biopropanols, or biodiesels) from any organic material,and can also comprise use of ultrasonic treatment of the biomassmaterial; see, e.g., U.S. Pat. No. 6,333,181.

In another aspect, methods of the invention for producing biofuels(including bioalcohols such as bioethanols, biomethanols, biobutanols orbiopropanols, or biodiesels) from a cellulosic substrate compriseproviding a reaction mixture in the form of a slurry comprisingcellulosic substrate, an enzyme of this invention and a fermentationagent (e.g., within a reaction vessel, such as a semi-continuouslysolids-fed bioreactor), and the reaction mixture is reacted underconditions sufficient to initiate and maintain a fermentation reaction(as described, e.g., in U.S. Pat. App. No. 20060014260). In one aspect,experiment or theoretical calculations can determine an optimum feedingfrequency. In one aspect, additional quantities of the cellulosicsubstrate and the enzyme are provided into the reaction vessel at aninterval(s) according to the optimized feeding frequency.

One exemplary process for making biofuels (including bioalcohols such asbioethanols, biomethanols, biobutanols or biopropanols, or biodiesels)of the invention is described in U.S. Pat. App. Pub. Nos. 20050069998;20020164730; and in one aspect comprises stages of grinding the anybiomass, e.g., an animal, algae and/or plant biomass includinglipid-comprising or lignocellulosic biomass (e.g., to a size of 15-30mm), subjecting the product obtained to steam explosion pre-treatment(e.g., at a temperature of 190-230° C.) for between 1 and 10 minutes ina reactor; collecting the pre-treated material in a cyclone or relatedproduct of manufacture; and separating the liquid and solid fractions byfiltration in a filter press, introducing the solid fraction in afermentation deposit and adding one or more enzymes of the invention,e.g., a cellulase and/or beta-glucosidase enzyme (e.g., dissolved incitrate buffer pH 4.8).

Another exemplary process for making biofuels (including bioalcoholssuch as bioethanols, biomethanols, biobutanols or biopropanols, orbiodiesels) of the invention comprising bioethanols, biomethanols,biobutanols or biopropanols using enzymes of the invention comprisespretreating a starting material comprising any biomass, e.g., an animal,algae and/or plant biomass including lipid-comprising or lignocellulosicbiomass feedstock comprising at least hemicellulose and cellulose. Inone aspect, the starting material comprises potatoes, soybean(rapeseed), barley, rye, corn, oats, wheat, beets or sugar cane or acomponent or waste or food or feed production byproduct. The startingmaterial (“feedstock”) is reacted at conditions which disrupt theplant's fiber structure to effect at least a partial hydrolysis of thehemicellulose and cellulose. Disruptive conditions can comprise, e.g.,subjecting the starting material to an average temperature of 180° C. to270° C. at pH 0.5 to 2.5 for a period of about 5 seconds to 60 minutes;or, temperature of 220° C. to 270° C., at pH 0.5 to 2.5 for a period of5 seconds to 120 seconds, or equivalent. This generates a feedstock withincreased accessibility to being digested by an enzyme, e.g., acellulase enzyme of the invention. U.S. Pat. No. 6,090,595.

Exemplary conditions for using enzymes of the invention in thehydrolysis of any biomass, e.g., an animal, algae and/or plant biomassincluding lipid-comprising or lignocellulosic biomass include reactionsat temperatures between about 30° C. and 48° C., and/or a pH betweenabout 4.0 and 6.0. Other exemplary conditions include a temperaturebetween about 30° C. and 60° C. and a pH between about 4.0 and 8.0.

Glucanases, (or cellulases), mannanases, xylanases, amylases,xanthanases and/or glycosidases, e.g., cellobiohydrolases, mannanasesand/or beta-glucosidases of the invention can be used in the conversionof biomass to fuels, and in the production of ethanol, e.g., asdescribed in PCT Application Nos. WO 0043496 and WO 8100857. Glucanases(or cellulases), mannanases, xylanases, amylases, xanthanases and/orglycosidases, e.g., cellobiohydrolases, mannanases and/orbeta-glucosidases of the invention can be used to produce fermentablesugars and glucan-containing biomass that can be converted into fuelethanol.

BioDiesels—Using Enzymes of the Invention to Make them

The invention provides compositions, including enzymes of the invention,and methods, for making biodiesel fuels, including any biofuel, e.g., abiodiesel, comprising alkyl esters made from the transesterification ofvegetable oils and/or animal fats.

For example, in alternative aspects, polypeptides of the invention,including the mixture of enzymes or “cocktails” of the invention, areused in processes for a transesterification process reacting an alcohol(like ethanol, propanol, butanol, propanol, methanol) with atriglyceride oil contained in a vegetable oil, animal fat or recycledgreases, forming fatty acid alkyl esters—including biodiesel—andglycerin. In one aspect, biodiesel is made from soybean oil or recycledcooking oils. Animal's fats, other vegetable oils, and other recycledoils can also be used (and processed by enzymes, e.g., phospholipases,of the invention) to produce a biodiesel, depending on their costs andavailability. In another aspect, blends of all kinds of fats and oilsare used to produce a biodiesel fuel of the invention using enzymes ofthe invention.

The invention provides compositions, including enzymes of the invention,and methods, for processing “yellow grease”, a term initially coined bythe rendering industry. Yellow grease that can be processed using thecompositions and methods of the invention include grease from fryingoils, e.g., from deep fryers or restaurants' grease traps, or fromvarious (e.g., lower-quality) grades of tallow from rendering plants.Thus, the invention also provides oils, grease, flying oils, vegetableoils, waste restaurant greases and processes grades of tallow comprisingat least one enzyme of this invention.

Yellow grease processed using compositions of the invention, includingenzymes, and methods of the invention, can be used to spray on roads,e.g., for dust control, or for animal feed additives or feeds, or foodsupplements.

In another aspect, compositions of the invention, including enzymes, andmethods of the invention, can be used to process lipids, e.g., greasessuch as waste restaurant greases to make a biofuel, e.g., a biodieselfuel, e.g., for cars, buses, trucks or boats. In one aspect, biodieselmade using a composition or method of the invention can be generatedfrom any renewable plant source, e.g., soybeans, and/or from a grease,such as the “yellow grease”.

Compositions of the invention, including enzymes, and methods of theinvention, can be used to process “SVO”, or “straight vegetable oil”,including any vegetable oil that can fuel a diesel engine, e.g., whereinthe processing comprises transesterification of lipids in the fuel,e.g., for use in lower temperatures.

Compositions of the invention, including enzymes, and methods of theinvention, can be used to process “WVO”, or waste vegetable oil, tomake, e.g., a yellow grease, including the grease from restaurants; inone aspect, the grease has to be filtered to remove food particles.Yellow grease processed by compositions of the invention, includingenzymes, and methods of the invention, can fall in the category ofSVO/WVO, including any grease, e.g., a restaurant waste grease, that cancontain beef tallow and other animal products.

Distillers Dried Grain Processing

In another aspect, the enzymes (e.g., phospholipases) of the inventioncan be used to treat/process “distillers dried solubles (DDS)”,“distillers dried grains (DDS)”, “condensed distillers solubles (CDS)”,“distillers wet grains (DWG)”, and “distillers dried grains withsolubles (DDGS)”; distillers dried grains can be a cereal byproduct of adistillation process, and can include solubles. These processes cancomprise dry-grinding plant by-products, e.g. for feed applications,e.g., for poultry, bovine, swine and other domestic animals. Thus, theenzymes of the invention can be used to treat/process grains, e.g.,cereals, that are byproducts of any distillation process, includingprocesses using any source of grain, for example, the traditionalsources from brewers, or alternatively, from an ethanol-producing plant(factory, mill or the like). Enzymes of the invention can be used totreat/process drying mash from distilleries; this mash can besubsequently used for a variety of purposes, e.g., as fodder forlivestock, especially ruminants; thus the invention provides methods forprocessing fodder for livestock such as ruminants, and enzyme-processedfodder comprising phytases of this invention.

Enzymes of this invention can be used alone or with other enzymes toprocess “distillers dried solubles (DDS)”, “distillers dried grains(DDS)”, “condensed distillers solubles (CDS)”, “distillers wet grains(DWG)”, and “distillers dried grains with solubles (DDGS)”. For example,enzymes of this invention can be used in any step of an alcohol productprocess as illustrated in FIG. 12. Enzymes of this invention can be usedto increase the bioavailability of phosphorus in any biofuel, orpotential biofuel, including phosphorus found in “distillers driedsolubles (DDS)”, “distillers dried grains (DDS)”, “condensed distillerssolubles (CDS)”, “distillers wet grains (DWG)”, and “distillers driedgrains with solubles (DDGS)” (see, e.g., C. Martinez Amezcua, 2004Poultry Science 83:971-976).

Spirit, or Drinkable Alcohol Production

Enzymes of this invention of this invention also can be used inprocessing distillers dried grains for alcohol production—alcohol as in“spirits”, e.g., beer or whiskey production (in addition to use inprocessing biomass for making biofuels). Enzymes of this invention ofthis invention can be used in ethanol plants, e.g. for processing grainssuch as corn. Distillers dried grains can be made by first grinding agrain (e.g., corn) to a coarse consistency and adding to hot water.After cooling, yeast is added and the mixture ferments for several daysto a week. The solids remaining after fermentation are the distillersgrains. Phytases of this invention of this invention can be used at anystep of this process.

Formulations

The invention provides novel formulations comprising enzymes of thisinvention, and formulations for phospholipases of the invention,including formulations which include the novel enzymes of the invention.The enzymes of the invention can be used or formulated alone or asmixture of phospholipases of the invention, or other phospholipases, orother enzymes such as xylanases, cellulases, proteases, lipases,amylases, or redox enzymes such as laccases, peroxidases, catalases,oxidases, or reductases. They can be used formulated in a solid formsuch as a powder, a lyophilized preparation, a granule, a tablet, a bar,a crystal, a capsule, a pill, a pellet, or in a liquid form such as inan aqueous solution, an aerosol, a gel, a paste, a slurry, anaqueous/oil emulsion, a cream, a capsule, or in a vesicular or micellarsuspension. The formulations of the invention can comprise any or acombination of the following ingredients: polyols such as a polyethyleneglycol, a polyvinylalcohol, a glycerol, a sugar such as a sucrose, asorbitol, a trehalose, a glucose, a fructose, a maltose, a mannose, agelling agent such as a guar gum, a carageenan, an alginate, a dextrans,a cellulosic derivative, a pectin, a salt such as a sodium chloride, asodium sulfate, an ammonium sulfate, a calcium chloride, a magnesiumchloride, a zinc chloride, a zinc sulfate, a salt of a fatty acid and afatty acid derivative, a metal chelator such as an EDTA, an EGTA, asodium citrate, an antimicrobial agent such as a fatty acid or a fattyacid derivative, a paraben, a sorbate, a benzoate, an additionalmodulating compound to block the impact of an enzyme such as a protease,a bulk proteins such as a BSA, a wheat hydrolysate, a borate compound,an amino acid or a peptide, an appropriate pH or temperature modulatingcompound, an emulsifier such as a non-ionic and/or an ionic detergent, aredox agent such as a cystine/cysteine, a glutathione, an oxidizedglutathione, a reduced or an antioxidant compound such as an ascorbicacid, or a dispersant.

Cross-linking and protein modification such as pegylation, fatty acidmodification, glycosylation can also be used to improve enzymestability.

Other Uses for the Phospholipases of the Invention

The phospholipases of the invention can also be used to study thephosphoinositide (PI) signaling system; in the diagnosis, prognosis anddevelopment of treatments for bipolar disorders (see, e.g., Pandey(2002) Neuropsychopharmacology 26:216-228); as antioxidants; as modifiedphospholipids; as foaming and gelation agents; to generate angiogeniclipids for vascularizing tissues; to identify phospholipase, e.g., PLA,PLB, PLC, PLD and/or patatin modulators (agonists or antagonists), e.g.,inhibitors for use as anti-neoplastics, anti-inflammatory and asanalgesic agents. They can be used to generate acidic phospholipids forcontrolling the bitter taste in food and pharmaceuticals. They can beused in fat purification. They can be used to identify peptidesinhibitors for the treatment of viral, inflammatory, allergic andcardiovascular diseases. They can be used to make vaccines. They can beused to make polyunsaturated fatty acid glycerides andphosphatidylglycerols.

The phospholipases of the invention, for example PLC enzymes, are usedto generate immunotoxins and various therapeutics for anti-cancertreatments.

The phospholipases of the invention can be used in conjunction withother enzymes for decoloring (i.e. chlorophyll removal) and indetergents (see above), e.g., in conjunction with other enzymes (e.g.,lipases, proteases, esterases, phosphatases). For example, in anyinstance where a PLC is used, a PLD and a phosphatase may be used incombination, to produce the same result as a PLC alone.

The following Table 7 summaries several exemplary processes andformulations of the invention:

TABLE 7 Purposes Exemplary Processes of the invention Chemical usage inPLC oil degumming No use of acid Chemical elimination No use of causticChemical elimination Range of acid and caustic use (no excess Chemicalreduction/degumming process to excess) alternative embodiment Othertypes of acid and caustic Degumming process alternative embodimentsImpact of water in PLC oil degumming Use of silica gel Replacement ofwater wash step Use of water drying agent Elimination of water in finalproduct Impact of lower water during caustic Elimination of water infinal product treatment Minimal water content (<5%) Elimination of waterin final product Maximal water content (>5%) Process alternativeHumidity profiles on PLC degumming Degumming process alternativeembodiment Oil dependence on water content for PLC Degumming processalternative embodiment degumming In situ removal of free fatty acids,FFAs Addition of FFA chelating agent Degumming process alternativeembodiment; improves conditions in oil from spoilt beans Impact ofmixing regimen on PLC oil degumming PLC degumming with minimal mixingProtection of enzyme from mixing induced denaturation, energy savingsPLC degumming with initial shear Degumming process alternativeembodiment mixing, followed by paddle mixing Order of addition ofchemicals Order of addition: enzyme-water followed Allow the PLC to workbefore exposure to by acid then caustic acid and or caustic, causingpotential pH or metal chelation PLC inactivation PLC oil degummingprocess alternative embodiments for temperature and time Enzymetreatment step (time): <60 min, Degumming process alternative embodimentpreferably <30 min Enzyme treatment step (temperature): 50-70° C.,Degumming process alternative embodiment possibly <50° C. (e.g. RT)Benefits from PLC oil degumming Producing soapstock with minimized PLDegumming process alternative embodiment content and enriched in watersoluble phosphate esters Reduced neutral oil in gum through use ofDegumming process alternative embodiment PLC Process of generatingincrease of DAG in Degumming process alternative embodiment vegetableoils (for ex, 1,3-DAG) Benefits of using increased DAG Exemplary Productbenefit vegetable oils with other oils for health benefits Investigatedegumming process that Degumming process alternative embodiment/ leavesno PLC activity in oil regulatory improvement Investigate degummingprocess that Degumming process alternative embodiment/ leaves nodetectable PLC protein in oil regulatory improvement Use of an enzyme toproduce DAG from Exemplary Product benefit lecithin gum mass Use of PLCwith specialty oils (PA, PI Exemplary Product benefit enriched) Use ofPA/PI specific enzymes (e.g. Degumming process alternative embodiment596ES2/PI specific) Use of PA/PI specific enzymes (e.g. Degummingprocess alternative embodiment 596ES2/PI specific) + PC/PE specificenzymes; impact of order of addition Batch or continuous processDegumming process alternative embodiment Use of resuspended PLC treatedgum for Degumming process alternative embodiment further oil degummingoperations Mass balance for DAG, FFA, P, metals, Degumming processalternative embodiment neutral oil in gum Miscellaneous Addition of PLCto flaked oil seed kernels Process alternative embodiment beforeextrusion Small scale degumming assay Degumming process alternativeembodiment Use of other enzymes to reduce gum mass Degumming processalternative embodiment (e.g., PYROLASE ® enzyme, chlorophyllase,peroxidase, lipase, laccase, mannanase, protease, lactase, amylase, etc.or combinations thereof) Use of compound to better facilitate Degummingprocess alternative embodiment oil/gum separation Harden gum from PLCtreated oil Degumming process alternative embodimentGlycosylated/deglycosylated variants of Degumming process alternativeembodiment phospholipase Exemplary Formulations of the inventionExemplary Liquid formulation for stability Use of compounds to increasethe stability Stabilization of enzyme for maximum DAG of PLC atdifferent pH and temp. ranges production, possibly for alteringsubstrate (polyols, salts, metals . . . ) specificity or directingproduct formation towards the 1,3-DAG type Use of a hydrophobic deliverysystem for Stabilization of enzyme for maximum DAG PLC (liposomes,hydrated enzyme in production, possibly for altering substrate refinedoil droplets) specificity or directing product formation towards the1,3-DAG type Solid formulation for stability Use of different PLC,phospholipase Stabilization of the enzyme(s) and ease of carrier systems(immobilization resins, separation of the enzyme from the oil or gumporous matrices, gels, granules, powders, phase after degumming;recyclability of the tablets, vesicles/micelles, encapsulates, enzymepreparation; physical separation of structured liquids, etc) tostabilize the enzyme phase during oil processing; phospholipase andco-enzymes attack of PI/PA by PLC Use of degumming waste materials (gumCost reduction of formulation ingredient, components, seed hulls) forPLC better miscibility of enzyme with oil, formulationthermostabilization of enzyme Exemplary Formulation and processes foractivity boost Use of chemical or enzyme to help Faster reactiontime/degumming disperse the enzyme better in oil (e.g. process/reductionof chemical usage effervescent matrix, etc) Re-use of gums/enzyme forfurther Recyclability of enzyme degumming reactions Use of formulationsthat enhance the Faster reaction time/degumming segregation or enzymecapture of PLs for process/reduction of chemical usage hydrolysis Use ofmultiple formulations to Versatility of process; different enzymes mayaccommodate PLCs with different PL require different formulations or maybe specificities added at different stages in the process Use ofmultiple formulations to prevent Protection of PLC activities in amulti- inactivation of one PLC by a component enzyme format embodimentin the prep of another PLC with a different substrate specificity Use ofmultiple formulations to prevent Protection of PLC activity in amulti-enzyme inactivation of one PLC by a component format embodiment inthe prep of another enzyme (hydrolase, oxidase) Use of intermittentcaustic additions as in Protection of enzyme from mixing induced timereleased caustic addition formulation denaturation, energy savingsInactivating and Modulating Activity of Enzymes by Glycosylation

This invention provides methods comprising use of recombinant technologyto make and expressing enzymes or other proteins with biologicalactivity, e.g., noxious or toxic enzymes, (wherein the enzymes or otherproteins are not normally glycosylated) in an inactive or less active,but re-activatable, form. The method comprises adding one or moreglycosylation sites (e.g., N-linked or O-linked glycosylation) into theenzymes or other proteins with biological activity (e.g., an enzyme ofthe present invention) by engineering a coding sequence incorporatingthe new glycosylation site(s); expressing the variant coding sequencesin eukaryotic cells or an equivalent engineered or in vitro systemcapable of post-translational glycosylation. For example, the 3 aminoacid sequence NXS/T is the site for glycosylation in eukaryotic cells,prokaryotic cells do not do this. Thus, the invention comprises addingat least one 3 amino acid sequence NXS/T to the protein such that itsactivity is decreased or inactivated because of post-translationalglycosylation.

The glycosylation can result in 2 molecules of N-acetyl glucosamine(NGlucNac) being added to the N residue. Subsequent additions can beorganism specific. In most species mannose (Mann) sugars are then addedonto the NGlucNac, with the number Mann residues ranging from 10 to 100.Sialic acid can also be added in some species. In Pichia after theNGlucNac is added, 10 to 25 Mann residues can be added.

These methods comprise using any deglycosylating enzyme or set ofenzymes, many of which can have been identified and/or are commerciallyavailable. For example, the endoglycosidase H enzyme cleaves at the lastNGlucNac leaving one NGlucNac still attached to the N residue. ThePNGaseF enzyme cleaves off all of the sugars and converts the amino sidechain of the N residue into a hydroxyl group resulting in the N aminoacid becoming an aspartate (D) amino acid in the enzyme. Thus, themethods comprise using endoglycosidase H and/or PNGaseF or equivalentenzymes in vivo or in vitro to re-activate partially or completely theengineered “temporarily inactivated” proteins.

The method comprises targeting the enzymes or other polypeptides to thehost secretory pathway so that the enzymes will be glycosylated. The newglycosylation sites are designed such that glycosylation inactivates theenzyme or modifies its activity, e.g., decreases it activity or otherotherwise modifies activity, such as blocks a substrate binding site.Because the enzyme is inactive or less active, noxious or toxic enzymescould be expressed at higher levels since the negative effects of theiractivity are no longer a limitation to how much of the protein canaccumulate in the host cells. The inactive, glycosylated enzyme can bere-activated (partially or completely) by removing the sugars, e.g.,using commercially available deglycosylating enzymes, for example, byremoving the sugars in vitro, or removing the sugars in vivo using wholecell engineering approaches.

In one aspect, a eukaryotic glycosylation target site such as NXS/T isadded to any protein, for example, an enzyme of the invention. Thisenables one skilled in the art to add glycosylation sites to a proteinof interest with the expectation of converting that protein into onethat is temporarily inactive when that protein is glycosylated byexpressing that protein in a eukaryotic host cell and targeting theprotein to the host cell's secretory pathway.

Thus, the invention provides methods for the production of enzymes thatnormally are too noxious or toxic to be tolerated in large amounts by ahost cell. The effect can temporary as it is possible to regenerate theactive enzyme (by deglycosylation, e.g., by post-translationalmodification/deglycosylation) for future work requiring an activeenzyme.

In one aspect, the invention provides methods for making and expressinga protein having a biological activity whose activity is temporarilyinactivated by glycosylation comprising: (a) providing a nucleic acidencoding a protein having a biological activity, wherein the protein isnot naturally glycosylated; (b) inserting at least one glycosylationmotif coding sequence into the protein-encoding nucleic acid, whereinthe glycosylated form of the protein is inactive; (c) inserting atargeting sequence into the protein such that it is directed to a hostcell's secretory pathway, wherein the host cell is capable ofrecognizing the glycosylation motif and glycosylating the protein; and(d) expressing the modified nucleic acid in the host cell. In oneaspect, the method further comprises deglycosylating the expressed theprotein, thereby re-activating the activity of the protein, e.g., anenzyme, such as an enzyme of the invention. In one aspect, the host cellis a eukaryotic cell. In one aspect, the inactivated expressedrecombinant protein is re-activated in vitro by deglycosylation, eitherchemical or enzymatic.

Determining the placement of one or more glycosylation motifs totemporarily inactivate a protein involves only routine methods of makingvariant protein-encoding nucleic acids, e.g., by GSSM, and routinescreening protocols, e.g., activity or binding assays.

An enzyme whose activity was detrimental to the host cell was renderedinactive because of glycosylation. Because it was inactive it couldaccumulate in much higher levels in the eukaryotic host cells. Becauseit was no longer active it could no longer able to exert its negativeeffects. The inactivation of the toxic enzyme was temporary becausedeglycosylating the enzyme using EndoH or PNGase F resulted in acomplete restoration of normal activity to the enzyme. A large amount ofthe glycosylated, inactive enzyme accumulated in the medium suggestingthat it was tolerated well by the host as the inactive form.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative, and are not to be takenas limitations upon the scope of the subject matter. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the methods of use provided herein, may bemade without departing from the spirit and scope thereof. Patents,patent publications, and other publications referenced herein areincorporated by reference.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Blast Program Used for Sequence Identify Profiling

This example describes an exemplary sequence identity program todetermine if a nucleic acid is within the scope of the invention. AnNCBI BLAST 2.2.2 program is used, default options to blastp. All defaultvalues were used except for the default filtering setting (i.e., allparameters set to default except filtering which is set to OFF); in itsplace a “-F F” setting is used, which disables filtering. Use of defaultfiltering often results in Karlin-Altschul violations due to shortlength of sequence. The default values used in this example:

 “Filter for low complexity: ON > Word Size: 3 > Matrix: Blosum62 > GapCosts: Existence:11 > Extension:1”

Other default settings were: filter for low complexity OFF, word size of3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gapextension penalty of −1. The “-W” option was set to default to 0. Thismeans that, if not set, the word size defaults to 3 for proteins and 11for nucleotides. The settings read:

<<README.bls.txt>> >-------------------------------------------------------------------------- >blastall arguments: > > -p Program Name [String] > -d Database[String] > default = nr > -i Query File [File In] > default = stdin > -eExpectation value (E) [Real] > default = 10.0 > -m alignment viewoptions: > 0 = pairwise, > 1 = query-anchored showing identities, > 2 =query-anchored no identities, > 3 = flat query-anchored, showidentities, > 4 = flat query-anchored, no identities, > 5 =query-anchored no identities and blunt ends, > 6 = flat query-anchored,no identities and blunt ends, > 7 = XML Blast output, > 8 = tabular, > 9tabular with comment lines [Integer] > default = 0 > -o BLAST reportOutput File [File Out] Optional > default = stdout > -F Filter querysequence (DUST with blastn, SEG with others) [String] > default = T > -GCost to open a gap (zero invokes default behavior) [Integer] > default =0 > -E Cost to extend a gap (zero invokes default behavior) [Integer] >default = 0 > -X X dropoff value for gapped alignment (in bits) (zeroinvokes default > behavior) [Integer] > default = 0 > -I Show GI's indeflines [T/F] > default = F > -q Penalty for a nucleotide mismatch(blastn only) [Integer] > default = −3 > -r Reward for a nucleotidematch (blastn only) [Integer] > default = 1 > -v Number of databasesequences to show one-line descriptions for (V) > [Integer] > default =500 > -b Number of database sequence to show alignments for (B)[Integer] > default = 250 > -f Threshold for extending hits, default ifzero [Integer] > default = 0 > -g Perform gapped alignment (notavailable with tblastx) [T/F] > default = T > -Q Query Genetic code touse [Integer] > default = 1 > -D DB Genetic code (for tblast[nx] only)[Integer] > default = 1 > -a Number of processors to use [Integer] >default = 1 > -O SeqAlign file [File Out] Optional > -J Believe thequery defline [T/F] > default = F > -M Matrix [String] > default =BLOSUM62 > -W Word size, default if zero [Integer] > default = 0 > -zEffective length of the database (use zero for the real size) >[String] > default = 0 > -K Number of best hits from a region to keep(off by default, if used a > value of 100 is recommended) [Integer] >default = 0 > -P 0 for multiple hits 1-pass, 1 for single hit 1-pass, 2for 2-pass > [Integer] > default = 0 > -Y Effective length of the searchspace (use zero for the real size) > [Real] > default = 0 > -S Querystrands to search against database (for blast[nx], and > tblastx). 3 isboth, 1 is top, 2 is bottom [Integer] > default = 3 > -T Produce HTMLoutput [T/F] > default = F > -l Restrict search of database to list ofGI's [String] Optional > -U Use lower case filtering of FASTA sequence[T/F] Optional > default = F > -y Dropoff (X) for blast extensions inbits (0.0 invokes default > behavior) [Real] > default = 0.0 > -Z Xdropoff value for final gapped alignment (in bits) [Integer] > default =0 > -R PSI-TBLASTN checkpoint file [File In] Optional > -n MegaBlastsearch [T/F] > default = F > -L Location on query sequence [String]Optional > -A Multiple Hits window size (zero for single hit algorithm)[Integer] > default = 40

Example 2 Modifications to a PLC Enzyme (ePLC)

This example describes exemplary protocols for making PLC enzymes ofthis invention, including PI-PLC enzymes of this invention. This exampledescribes enzymes that can be used to practice this invention, e.g.,used in combination with PLC enzymes of this invention (e.g., an enzymehaving a sequence as set forth in SEQ ID NO:8, or as described in Table12 to 15). Enzymes that can be used to practice this invention, e.g., incombinations or mixtures comprising PLC enzymes of this invention,include any phospholipase enzyme, including an enzyme having a sequenceas set forth in Table 8 or Table 9, or described in WO 2008/036863. Inalternative embodiments, enzymes that can be used to practice thisinvention include polypeptides having a sequence as set forth in SEQ IDNO:2 and/or SEQ ID NO:4, and variants thereof as described in Tables 8and 9, below.

Phospholipase C enzyme having a sequence as set forth in SEQ ID NO:2(encoded e.g. by SEQ ID NO:1) is an enzymatically active subsequence ofthe longer sequence SEQ ID NO:4 (encoded e.g. by SEQ ID NO:3). SEQ IDNO:4 has a leader sequence of residues 1 to 37 (bolded) of SEQ ID NO:2.SEQ ID NO:4, as encoded by SEQ ID NO:3, was used as a template forfurther modification using GSSM technology. Positions are numberedstarting with the N-terminal Methionine. Mutations are underlined and inbold (numbered here as N100D, N168S and N171D).

(SEQ ID NO: 4) MKKKVLALAA MVALAAPVQS VVFAQTNNSE SPAPILRWSAEDKHNEGINS HLWIVNRAID IMSRNTTIVN PNETALLNEW RADLENGIYS ADYENPYYD D STYASHFYDP DTGTTYIPFA KHAKETGAKY FNLAGQAYQN QDMQQAFFYL GLSLHYLGDVNQPMHAA S FT  D LSYPMGFHS KYENFVDTIK NNYIVSDSNGYWNWKGANPE DWIEGAAVAA KQDYPGVVND TTKDWFVKAAVSQEYADKWR AEVTPVTGKR LMEAQRVTAG YIHLWFDTYV NR (SEQ ID NO: 2)                             WSA EDKHNEGINSHLWIVNRAID IMSRNTTIVN PNETALLNEW RADLENGIYS ADYENPYYD D STYASHFYDP DTGTTYIPFA KHAKETGAKYFNLAGQAYQN QDMQQAFFYL GLSLHYLGDV NQPMHAA S FT DLSYPMGFHS KYENFVDTIK NNYIVSDSNG YWNWKGANPEDWIEGAAVAA KQDYPGVVND TTKDWFVKAA VSQEYADKWRAEVTPVTGKR LMEAQRVTAG YIHLWFDTYV NR (SEQ ID NO: 3)ATGAAAAAGAAAGTATTAGCACTAGCAGCTATGGTTGCTTTAGCTGCGCCAGTTCAAAGTGTAGTATTTGCACAAACAAATAATAGTGAAAGTCCTGCACCGATTTTAAGATGGTCAGCTGAGGATAAGCATAATGAGGGGATTAACTCTCATTTGTGGATTGTAAATCGTGCAATTGACATCATGTCTCGTAATACAACGATTGTGAATCCGAATGAAACTGCATTATTAAATGAGTGGCGTGCTGATTTAGAAAATGGTATTTATTCTGCTGATTACGAGAATCCTTATTATGATGATAGTACATATGCTTCTCACTTTTATGATCCGGATACTGGAACAACATATATTCCTTTTGCGAAACATGCAAAAGAAACAGGCGCAAAATATTTTAACCTTGCTGGTCAAGCATACCAAAATCAAGATATGCAGCAAGCATTCTTCTACTTAGGATTATCGCTTCATTATTTAGGAGATGTGAATCAGCCAATGCATGCAGCATCTTTTACGGATCTTTCTTATCCAATGGGTTTCCATTCTAAATACGAAAATTTTGTTGATACAATAAAAAATAACTATATTGTTTCAGATAGCAATGGATATTGGAATTGGAAAGGAGCAAACCCAGAAGATTGGATTGAAGGAGCAGCGGTAGCAGCTAAACAAGATTATCCTGGCGTTGTGAACGATACGACAAAAGATTGGTTTGTAAAAGCAGCCGTATCTCAAGAATATGCAGATAAATGGCGTGCGGAAGTAACACCGGTGACAGGAAAGCGTTTAATGGAAGCGCAGCGCGTTACAGCTGGTTATATTCATTTGTGGTTTGATAC GTATGTAAATCGCTAA(SEQ ID NO: 1) TGGTCAGCTGAGGATAAGCATAATGAGGGGATTAACTCTCATTTGTGGATTGTAAATCGTGCAATTGACATCATGTCTCGTAATACAACGATTGTGAATCCGAATGAAACTGCATTATTAAATGAGTGGCGTGCTGATTTAGAAAATGGTATTTATTCTGCTGATTACGAGAATCCTTATTATGATGATAGTACATATGCTTCTCACTTTTATGATCCGGATACTGGAACAACATATATTCCTTTTGCGAAACATGCAAAAGAAACAGGCGCAAAATATTTTAACCTTGCTGGTCAAGCATACCAAAATCAAGATATGCAGCAAGCATTCTTCTACTTAGGATTATCGCTTCATTATTTAGGAGATGTGAATCAGCCAATGCATGCAGCATCTTTTACGGATCTTTCTTATCCAATGGGTTTCCATTCTAAATACGAAAATTTTGTTGATACAATAAAAAATAACTATATTGTTTCAGATAGCAATGGATATTGGAATTGGAAAGGAGCAAACCCAGAAGATTGGATTGAAGGAGCAGCGGTAGCAGCTAAACAAGATTATCCTGGCGTTGTGAACGATACGACAAAAGATTGGTTTGTAAAAGCAGCCGTATCTCAAGAATATGCAGATAAATGGCGTGCGGAAGTAACACCGGTGACAGGAAAGCGTTTAATGGAAGCGCAGCGCGTTACAGCTGGTTATATTCATTTGTGGTTTGATACGTATGTAAATCGC TAA

Single-residue mutations were made using Gene Site SaturationMutagenesis (GSSM) methods described above and assayed for phospholipaseactivity. For screening purposes, the expression vector was pASK in E.coli host Top10. GSSM hits were selected from a primary screen for whicha PA/PI emulsion was used as the substrate and the samples were analyzedby LCMS. These primary hits were then confirmed on soybean oil andanalyzed by ³¹P NMR and HPLC.

The soybean oil assay and procedure for preparing the samples foranalysis by NMR is as follows:

NMR Detergent was made by dissolving 25 g Deoxycholic acid, 5.84 g EDTA,5.45 g Tris base in 900 mL of water, then adjust the pH to 10.5 usingKOH pellets. The internal NMR standard was 50 mM TIP and 12.5 mM TBP inHPLC-grade isopropanol. Deuterium oxide (D, 99.9%) low paramagnetic wasfrom Cambridge Isotope Laboratories Inc. (DLM-11-100). The NMR controlwas Avanti Lecithin (International Lecithin & Phospholipids Societymixed soy phospholipids reference Standard oil), Avanti Polar LipidsInc, #95309.

The standards and samples were prepared as follows:

-   -   Thoroughly mix a batch of Crude Soybean Oil    -   Dispense 1 mL of oil into a 2 mL tube. Add 60 μL of purified        enzyme (for controls, 18 Units) or pure cell lysate (for        screening mutants); mix for 15 seconds.    -   Units are defined as hydrolysis of 1 μmol PC per minute at        37° C. at pH 7.3.    -   Incubate at 60° C. for 48 hours in thermomixer shaking at 14000        rpm, vortexing intermittently.    -   After incubation, mix the samples thoroughly using a vortex    -   Weigh out 250 mg (+/−0.2 mg) of each sample into a 2 mL tube and        weigh out a    -   NMR control of 10 mg (+/−0.1 mg) of Avanti Lecithin.    -   Add 900 μL of NMR Detergent then add 100 μL of D₂O to each        sample.    -   Mix the samples thoroughly by vortexing and shaking in Eppendorf    -   Thermomixer, at 30-37° C. and 14000 rpm for 30 minutes    -   Centrifuge at 13,000 RPM for 10 minutes    -   Carefully remove the top oily layer    -   Add 750 μL of hexane to each sample and vortex gently*    -   Centrifuge at 13,000 RPM for 10 minutes    -   Carefully remove 600 μL of bottom aqueous layer and transfer to        a new tube    -   Add 25 μL of Internal Standard, mix well    -   Transfer 500 μL to a 5 mm NMR tube.

Release of DAG was measured by quantitative HPLC according to thefollowing protocol:

The sample solution was approximately 50 μl oil samples and 950 ulhexane/isopropanol (9:1) to make 1 ml. The standard solutions were, forexample 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, and 4 mg/ml of ENOVA™oil (Kao Corporation, Itasca, Ill.). Enova oil is high-DAG oil that hasa fatty acids distribution similar to regular vegetable oil (1,3-DAG and1,2-DAG).

HPLC Settings:

Column: Chromegasphere™ SI-60, 15 cm×4.6 mm

Temperature: 40° C.

Flow Rate: 2 mL/min

Injection volume: 20 ul

Mobile phase A: Hexane

Mobile phase B: Hexane/Isopropanol/Ethyl Acetate/Formicacid=800:100:100:1

Gradient elution:

Time (min) 0 8 8.5 15 15.1 19 % B 2 35 98 98 2 2

Evaporative Light Scattering Detector (ELSD) Settings:

A exemplary setting was temperature 40° C., gain 5, and nitrogen gas 3.5bars. The DAG peak was identified by comparison of retention time withthat of standard. Quantification was based on the relationship betweenthe detector's response (peak area) and the analyte's concentration.

Table 8 describes sequences that can be used to practice this invention,e.g., in combination with polypeptides of this invention (see, e.g.,Tables 12 to 15), e.g., as mixtures or combinations of enzymes.

Based on: NMR and HPLC data, the mutations shown in Table 8, below, wereselected. Table 8, below, indicates the starting amino acid, theposition number of the amino acid change and the changed amino acid (forSEQ ID NO:4). Table 8 also indicates the original codon, the replacementcodon and other codons for the same changed to amino acid. For example,the second row, “E41A”, indicates that the amino acid in position 41 wasoriginally “E” (glutamic acid), but was changed to “A” (alanine). Theoriginal codon for change E41A was “GAG”, but was changed to “GCA”.However, codons “GCG”, “GCC” or “GCT” could also have been used. Thecodon variants as set forth in Table 8 that produced variants (of SEQ IDNO:4) with the best variation or “improvement” over “wild type” (SEQ IDNO:4) for PA hydrolysis. The invention provides nucleic acids, and thepolypeptides that encode them, comprising one, several or all or thevariations, or the equivalent of all the variations, set forth in Table8.

In FIG. 10, the weight-fraction of individual phospholipid (PL) speciesis given relative to the total PL remaining after the reaction,reflecting the specificity of the mutants to particular species. Here,the species were phosphatidic acid (PA), phosphatidylethanolamine (PE),phosphatidylinositol (PI), phosphatidylcholine (PC). “TIP” refers to theinternal NMR standard. “DAG released” was measured by HPLC and reflectsrelative values between samples and controls of total 1,3-DAG and1,2-DAG. The positive control was a purified sample of E41A mutantpreviously described in Tan et al., Biochemistry 37:4275-4279 (1998).The results indicate that the mutants release DAG well and have goodactivity on various species, including phosphatidylcholine (PC) andphosphatidylethanolamine (PE), comparable or better than the template(SEQ ID NO:4). For example D100L and D100M show particular activity onPA. Q265R shows particular activity on PI. These mutations can becombined to provide enzymes having desired activities on varioussubstrates.

TABLE 8 GSSM hits PLC Other codons encoding AA Codon GSSM OriginalChanged the same “changed to” Original Changed Mutation Hits Codon To AAAA To Location E41A GAG GCA GCG, GCC, GCT E A  41 E41W GAG TGG — E W  41E41F GAG TTC TTT E F  41 E41Y GAG TAC TAT E Y  41 E41R GAG CGTCGC, CGA, CGG, AGA, E R  41 AGG E94R GAG CGG CGC, CGA, CGT, AGA, E R  94AGG D100L GAT TTG CTC, TTA, CTT, CTA, D L 100 CTG D100M GAT ATG — D M100 D100Y GAT TAT TAC D Y 100 D100F GAT TTT TTC D F 100 D100W GAT TGG —D W 100 A104L GCT CTT CTC, TTA, TTG, CTA, A L 104 CTG D111R GAT AGGCGC, CGA, CGT, AGA, D R 111 CGG T112R ACT CGG CGC, CGA, CGT, AGA, T R112 AGG Y116W TAT TGG — Y W 116 I117W ATT TGG — I W 117 P118W CCT TGG —P W 118 E125K GAA AAG AAA E K 125 S168N TCT AAC AAT N S 168 D171V GATGTG GTT, GTC, GTA D V 171 D171E GAT GAG GAA D E 171 M176W ATG TGG — M W176 D230H GAT CAT CAC D H 230 D230R GAT CGT CGC, CGA, CGG, AGA, D R 230AGG D234W GAT TGG — D W 234 D234V GAT GTG GTT, GTC, GTA D V 234 D234GGAT GGT GGC, GGA, GGG D G 234 D234R GAT CGG CGC, CGA, CGT, AGA, D R 234AGG D234K GAT AAG AAA D K 234 Q265R CAG CGT CGC, CGA, CGG, AGA, Q R 265AGG

In alternative embodiments, the invention provides combinations(mixtures) of PLC enzymes, or the nucleic acids that encode them,comprising the nucleic acid sequence SEQ ID NO:3 (encoding thepolypeptide SEQ ID NO:4) and/or the nucleic acid sequence SEQ ID NO:1(encoding the polypeptide SEQ ID NO:2); or combinations (mixtures) ofPLC enzymes comprising SEQ ID NO:2 and/or SEQ ID NO:4.

In alternative embodiments, the invention provides combinations(mixtures) of PLC enzymes, or the nucleic acids that encode them,comprising the nucleic acid sequence SEQ ID NO:3 (encoding thepolypeptide SEQ ID NO:4) and/or the nucleic acid sequence SEQ ID NO:1(encoding the polypeptide SEQ ID NO:2) having one, two, or more or allof nucleic-acid (mutations) that encode the amino-acid mutations listedabove in Table 8, including e.g. the codon changes described herein. Inalternative embodiments, the invention provides combinations (mixtures)of PLC enzymes encoded by these nucleic acids, e.g., combinations(mixtures) of PLC enzymes encoded by one, several or all of the nucleicacid sequence variations of SEQ ID NO:3 and/or SEQ ID NO:1, as describedin Table 8.

After GSSM hits were screened and the top hits selected (see Table 8,above), further characterization assays on eggyolk plates were performedin order to narrow down the number of single GSSM mutants carried forthfor combination using GeneReassembly technology. Table 10 shows theeggyolk assay data (eggyolk assay described below), along with theresults of oil assays and thermal tolerance residual activitydetermination. FIG. 11 illustrates the single GSSM upmutants that wereselected for inclusion in the GeneReassembly process. GeneReassembly wasperformed as described herein.

Table 9, below, lists 288 polypeptide sequences that can be used topractice this invention, e.g., in combination with polypeptides of thisinvention (see, e.g., Tables 12 to 15), e.g., as mixtures orcombinations of enzymes. The Table 9 sequences were created byGeneReassembly combination of the selected single GSSM upmutants. Allare variants of the starting amino acid sequence SEQ ID NO:4 (the “wildtype” or “WT” sequence).

To aid in reading Table 9, for example, for the phospholipasecharacterized as “evolved” phospholipase 1 (second row of table):

-   -   the wild-type amino acid residue “E”, or glutamic acid (glu) at        residue position 41 (of SEQ ID NO:4) is modified to a “Y”, or        tyrosine (tyr) residue;    -   the wild-type amino acid residue “N”, or asparagine (asp) at        residue position 100 (of SEQ ID NO:4) is modified to a “M”, or        methionine (met) residue;    -   the wild-type amino acid residue “N”, or asparagine (asp) at        residue position 168 (of SEQ ID NO:4) is modified to an “S” or        serine (ser) residue;    -   the wild-type amino acid residue “N”, or asparagine (asp) at        residue position 171 (of SEQ ID NO:4) remains an “N”; and,    -   the wild-type amino acid residue “M”, or methionine (met) at        residue position 176 (of SEQ ID NO:4) remains an “M”.

TABLE 9 Phospholipase Library Resulting from GeneReassembly Combinationof Single GSSM Upmutants “Evolved” Phospholipase E41 N100 N168 N171 M1761 Y M S N M 2 F W S E M 3 A M N E W 4 Y F S E M 5 Y Y S N M 6 R F N E M7 E Y N E M 8 E F N N W 9 A W S E M 10 Y Y S N W 11 E L S N W 12 A F N NM 13 W M N N M 14 W Y S E M 15 R L N E W 16 W W S E W 17 W N S N M 18 WL N E M 19 R N N E M 20 F N N N W 21 Y N S E M 22 R N S N W 23 F Y S N W24 F L N E W 25 A N N E M 26 A W N N M 27 W M N E W 28 F L S E W 29 Y FS N M 30 F F N N M 31 E W N E M 32 E W N N W 33 E W S E M 34 E Y S N M35 E N S N M 36 E L N E W 37 Y M N E M 38 F N S N W 39 W N N E W 40 E MS N M 41 Y N S N M 42 Y Y N E M 43 Y L N E M 44 F M N N W 45 F N S E M46 F M S N W 47 E F S N W 48 W Y N E W 49 F F N E M 50 R M S N W 51 A NN E W 52 R W S N M 53 R L S N M 54 R W N E M 55 F W N N M 56 E L N N W57 E L S E M 58 A Y S N W 59 E Y S N W 60 W N N E M 61 W N N N W 62 A FS N M 63 Y M S E W 64 R F S N M 65 A M N N M 66 F N N E M 67 E M N E M68 E Y S E M 69 E F S E M 70 E W S N M 71 F W S N W 72 E W N E W 73 Y LN N W 74 Y N S N W 75 A Y S E W 76 E F S N M 77 W L S N M 78 Y N N E M79 E F N E M 80 W N S E M 81 E M S E M 82 W N S N W 83 E W S N W 84 Y MN E W 85 E Y N N W 86 F M N E W 87 R L S E W 88 W F S N M 89 E L S N M90 E L N E M 91 Y F N N W 92 Y L S E M 93 A N S N W 94 E N N N W 95 E MS N W 96 R N N E W 97 E M N E W 98 F W S E W 99 W W N N M 100 W N N N M101 E N S E W 102 R W S E W 103 A W S E W 104 A Y S E M 105 F Y S E W106 A Y N N W 107 R N S N M 108 F F N N W 109 Y N N E W 110 E W S E W111 R N S E M 112 E L N N M 113 E N S N W 114 R W S N W 115 F W N E M116 Y Y N E W 117 F Y N N W 118 W Y S N W 119 A N S N M 120 A L S N W121 E Y N E W 122 E Y S E W 123 W N S E W 124 E M N N M 125 E N N E M126 Y W N E W 127 A W N N W 128 Y Y S E M 129 W Y N N W 130 F Y N E M131 A N S E M 132 A L S E W 133 E F N E W 134 R N S E W 135 F N S E W136 E W N N M 137 E N N E W 138 W W N E W 139 Y W S N W 140 W Y N E M141 R Y S N W 142 F Y S N M 143 Y F S N W 144 R L N E M 145 F N N E W146 Y N S E W 147 R N N N M 148 E Y N N M 149 R W S E M 150 Y W N N W151 A W S N W 152 R Y S E M 153 R Y N E M 154 W Y S E W 155 A Y N E W156 Y M N N W 157 Y F N N M 158 A N S E W 159 Y N N N M 160 E F N N M161 Y W S E M 162 Y W N E M 163 W W N N W 164 F Y N E W 165 W Y S N M166 A Y N E M 167 F F S E W 168 W L S E M 169 Y Y S E W 170 E L S E W171 F N N N M 172 E N N N M 173 W W S E M 174 A W N E M 175 R Y N N W176 A Y S N M 177 R Y S N M 178 R Y S E W 179 R M N E W 180 W F N E M181 E F S E W 182 E M S E W 183 A N N N M 184 E N S E M 185 F W N N W186 F W S N M 187 R Y N E W 188 Y Y N N W 189 F Y S E M 190 F N S N M191 R F S E W 192 F L S N W 193 W Y N N M 194 A L N N M 195 F L N N M196 A F S E W 197 W F S E W 198 A F N E W 199 R L S N W 200 W L N E W201 Y L S N W 202 R M N E M 203 A M S E W 204 Y W S N M 205 R Y N N M206 Y M N N M 207 W M N N W 208 F F S E M 209 Y F S E W 210 W F S E M211 W L S N W 212 R L N N W 213 W L N N W 214 W M S N W 215 W M S E W216 R W N E W 217 R L N N M 218 R F N N M 219 A N N N W 220 Y F N E M221 W F N N W 222 R F S E M 223 F L S N M 224 F L S E M 225 A M S E M226 A M S N M 227 R M S E M 228 R W N N W 229 A Y N N M 230 Y W N N M231 E M N N W 232 A F S E M 233 W F N E W 234 W F S N W 235 A L S E M236 Y L N E W 237 F M S E M 238 W M S E M 239 F M S E W 240 Y W S E W241 W F N N M 242 W L N N M 243 R M N N W 244 R F N N W 245 A F N N W246 F F N E W 247 A L N E W 248 Y L S N M 249 F M S N M 250 Y M S E M251 F M N E M 252 F W N E W 253 Y L N N M 254 R W N N M 255 Y N N N W256 R F S N W 257 A F S N W 258 A F N E M 259 A L N N W 260 A L N E M261 R M S E W 262 W M S N M 263 R M S N M 264 A W S N M 265 R M N N M266 F Y N N M 267 A M N N W 268 F F S N M 269 Y F N E W 270 F L N E M271 Y L S E W 272 F L N N W 273 Y M S N W 274 A M N E M 275 A W N E W276 W W S N W 277 F M N N M 278 Y Y N N M 279 R N N N W 280 F F S N W281 R F N E W 282 A L S N M 283 W L S E W 284 R L S E M 285 W M N E M286 A M S N W 287 W W N E M 288 W W S N M

Table 10 summarizes the results of assays analyzing various enzymaticactivity, and expression system behavior, of exemplary enzymes of theinvention (and in the case of the Pichia Pastoris Expression system—theexpression activity of the nucleic acids that encode them), all of thepolypeptides of the invention being sequence variants of startingphospholipase sequence SEQ ID NO:4 (encoded, e.g., by the nucleic acidsequence SEQ ID NO:3).

TABLE 10 ACTIVITY ANALYSIS AND SUMMARY Pichia Pastoris Thermal ToleranceOil Assay Expression Percent Residual activity GSSM Upmutant GSSM % PAActivity on E. coli Pichia pastoris PLC GSSM Amino acid Amino AcidHydrolysis eggyolk Expressed Expressed Upmutants residue # change at 24hrs plates protein protein Crude oil 0 E41E 41 Wild type 20 Active 81%100% E41A 41 A 29 Active 83%  99% E41W 41 W 31 Active 94% N/A E41F 41 F68 Inactive 80% N/A E41Y 41 Y 69 Inactive 89% N/A E41R 41 R 66 Active78% 104% E94R 94 R 23 Active N/A N/A D100L 100 L 45 Active N/A  87%D100M 100 M 48 Active N/A 104% D100Y 100 Y 57 Active N/A 105% D100F 100F 59 Active 43%  92% D100W 100 W 61 Active N/A  91% A104L 104 L 26Active 115%   86% D111R 111 R 27 Active N/A  99% T112R 112 R 23 Active107%   92% Y116W 116 W 23 Active 118%  102% I117W 117 W 15 Active 109% 102% P118W 118 W 17 Active N/A N/A E125K 125 K 15 Active 99%  86% D171V171 V 29 Active N/A 106% D171E 171 E 44 Active N/A 110% M176W 176 W 42Active 101%  101% D230H 230 H 21 Active N/A  97% D230R 230 R 14 Active107%  104% D234W 234 W 10 Active 101%   98% D234V 234 V 0 Active 109% 102% D234G 234 G 3 Active 109%  114% D234R 234 R 27 Active 114%   90%D234K 234 K 23 Active N/A 101% Q265R 265 R 0 Inactive N/A N/A E41A NNN41, 100, 168, 171 A, N, N, N 72 63% E41A NKN 41, 100, 168, 171 A, N, K,N 75 65% E41A NRN 41, 100, 168, 171 A, N, R, N 79 75% E41A NSN 41, 100,168, 171 A, N, S, N 72 85%

Egg Yolk Assay

The egg yolk assay is performed as follows:

-   -   Egg yolk agar plates are prepared by adding 0.5% (by wt.) egg        yolk phosphatidylcholine to media prior to autoclaving. The        plates are more uniform if the phosphatidylcholine is dispersed        with a high shear mixer prior to autoclaving the media.    -   Wells are punched in the agar and equal volumes (for example,        2 ml) of serial dilutions of samples, including positive        control, are loaded in the wells.    -   The plates are left for 3-12 hours at 37° C., during which time        the enzyme diffuses out of the wells, hydrolyses the egg yolk        lecithin and forms precipitation zones due to the formation of        diacylglycerol.    -   The area within the precipitation ring, measured as ring        diameter or integrated density value (IDV) is plotted against        the standard curve for the positive control to determine the        activity of the sample phospholipase. The whole process can be        used to determine the unknown PLC activity of a sample. The        method is semi-quantitative.    -   Phosphatidylcholine (PC): From Sigma, Catalog No. P 5394    -   PC from Dried Egg Yolk, Type X-E, approx. 60% PC by TLC.

In alternative embodiments, the invention provides combinations ormixtures of enzymes of the invention and enzymes as described in Example2, including e.g., all of the enzyme variants described in Table 8 andTable 9, and in WO 2008/036863.

Example 3 Making Exemplary Phosphatidylinositol-Specific Phospholipase C(PI-PLC) Enzymes of the Invention

This example describes exemplary phosphatidylinositol-specificphospholipase C (PI-PLC) enzymes of the invention, including thepolypeptide having the sequence as set forth in SEQ ID NO:8, and thepolypeptides having a PI-PLC activity as described in Tables 12 to 15;and exemplary methods for making and using them, and assays fordetermining their phospholipase activity.

In alternative embodiments, the invention provides polypeptides having aPI-PLC enzyme activity. In some embodiments, these polypeptides wereconstructed by the following methods:

For this series of embodiments, the polypeptide having the amino acidsequence of SEQ ID NO:6 (encoded e.g., by SEQ ID NO:5) was selected asthe “parent” or “wild-type” sequence for further modification (or“evolution”); in particular, the underlined (see below) subsequence ofSEQ ID NO:6 (or SEQ ID NO:5) was used with the addition of a startingMethionine (e.g. MASSINV . . . ), as the “parent” or starting sequencefor “evolution” or sequence changes to make enzyme variants. Note the“parent” or starting sequence for “evolution” lacks the first 30 aminoacids, which includes the signal sequence (italics), or

MNNKKFILKLFICSMVLSAFVF, encoded e.g., by:ATGAACAATAAGAAGTTTATTTTGAAGTTATTCATATGTAGTATGGTACT TAGCGCCTTTGTATTT

The “parent” or starting sequence for “evolution” also lacks a predictedcleavage site (bold italics) GCTTTC (nucleic acid) or AF (amino acidresidues).

“Evolution” (sequence change, or “mutation”) was performed using “GeneSite Saturation Mutagenesis” (GSSM) and GeneReassembly (see above fordescription of GSSM and GeneReassembly) on SEQ ID NO:5 using theunderlined sequences, below, with the addition of nucleic acid encodinga starting “M” or methionine (e.g., for the encoded amino acid sequence,MASSINV . . . ), as the parent or starting sequence for “evolution”:

SEQ ID NO: 5:ATGAACAATAAGAAGTTTATTTTGAAGTTATTCATATGTAGTATGGTACTTAGCGCCTTT GTATTT

CAATGATAAGAAAACCGTTGCAGCTAGCTCTATTAATGTGCTTGAAAATTGGTCTAGATGGATGAAACCTATAAATGATGACATACCGTTAGCACGAATTTCAATTCCAGGAACACATGATAGTGGAACGTTCAAGTTGCAAAATCCGATAAAGCAAGTGTGGGGAATGACGCAAGAATATGATTTTCGTTATCAAATGGATCATGGAGCTAGAATTTTTGATATAAGAGGGCGTTTAACAGATGATAATACGATAGTTCTTCATCATGGGCCATTATATCTTTATGTAACACTGCACGAATTTATAAACGAAGCGAAACAATTTTTAAAAGATAATCCAAGTGAAACGATTATTATGTCTTTAAAAAAAGAGTATGAGGATATGAAAGGGGCGGAAAGCTCATTTAGTAGTACGTTTGAGAAAAATTATTTTCGTGATCCAATCTTTTTAAAAACAGAAGGGAATATAAAGCTTGGAGATGCTCGTGGGAAAATTGTATTACTAAAAAGATATAGTGGTAGTAATGAATCTGGGGGATATAATAATTTCTATTGGCCAGACAATGAGACGTTTACCTCAACTATAAATCAAAATGTAAATGTAACAGTACAAGATAAATATAAAGTGAGTTATGATGAGAAAATAAACGCTATTAAAGATACATTAAATGAAACGATTAACAATAGTGAAGATGTTAATCATCTATATATTAATTTTACAAGCTTGTCTTCTGGTGGTACAGCATGGAATAGTCCATATTATTATGCGTCCTACATAAATCCTGAAATTGCAAATTATATGAAGCAAAAGAATCCTACGAGAGTGGGCTGGATAATACAAGATTATATAAATGAAAAATGGTCACCATTACTTTATCAAGAAGTTATAAGAGCGAATAAGTCACTTGTAAAATAG SEQ ID NO: 6: MNNKKFILKLFICSMVLSAFVF

NDKKTVA ASSINVLENWSRWMKPINDDIPLARISIPGTHDSGTFKLQNPIKQVWGMTQEYDFRYQMDHGARIFDIRGRLTDDNTIVLHHGPLYLYVTLHEFINEAKQFLKDNPSETIIMSLKKEYEDMKGAESSFSSTFEKNYFRDPIFLKTEGNIKLGDARGKIVLLKRYSGSNESGGYNNFYWPDNETFTSTINQNVNVTVQDKYKVSYDEKINAIKDTLNETINNSEDVNHLYINFTSLSSGGTAWNSPYYYASYINPEIANYMKQKNPTRVGWIIQDYINEKWSPLLYQEVIRANKSLVK

Thus, the starting sequence for GSSM was a nucleic acid encoding:

MASSINVLENWSRWMKPINDDIPLARISIPGTHDSGTFKLQNPIKQVWGMTQEYDFRYQMDHGARIFDIRGRLTDDNTIVLHHGPLYLYVTLHEFINEAKQFLKDNPSETIIMSLKKEYEDMKGAESSFSSTFEKNYFRDPIFLKTEGNIKLGDARGKIVLLKRYSGSNESGGYNNFYWPDNETFTSTINQNVNVTVQDKYKVSYDEKINAIKDTLNETINNSEDVNHLYINFTSLSSGGTAWNSPYYYASYINPEIANYMKQKNPTRVGWIIQDYINEKWSPLLYQEVIRANKSLVK

“Evolved” nucleic acid variants (the new nucleic acid sequences made bysubjecting SEQ ID NO:5 to GSSM) were subcloned for expression in eitherE. coli (for the GSSM phase) or in P. fluorescens (for theGeneReassembly phase).

GSSM was performed as described in e.g. U.S. Pat. Nos. 6,171,820;6,238,884 (see also explanation herein). See also WO 2008/036863.

Resulting new “variant” or “evolved” nucleic acid and polypeptidesequences were assayed using a high throughput assay as follows:

SOP for High Throughput Thermal Stability Assay

The GSSM screens used the E. coli host, XL1Blue (Stratagene, San Diego,Calif.), with the pASK vector (IBA GmbH, Gottingen, Germany). TheGeneReassembly screens used the Pseudomonas fluorescens host (Dow GlobalTechnologies Inc., US Patent PUB. APP. NO. 20050130160, US Patent PUB.APP. NO. 20050186666 and US Patent PUB. APP. NO. 20060110747) with thepDOW1169 vector (Dow Global Technologies Inc., US Patent PUB. APP. NO.20080058262) and were selected by growth in M9 minimal medium (DowGlobal Technologies Inc., US Patent PUB. APP. NO. 20050186666).

Master Plates

-   -   1. The Master Plates were created by colony picking GSSM or        GeneReassembly variants into a 384 well plate containing 50 μL        of media per well.        -   a. The media used for growing the GSSM variants was LB and            the GeneReassembly variants used M9 (-uracil).    -   2. Master plates were grown overnight at 30° C. in a humidified        incubator. Followed by the addition of 20% glycerol before        storing the plates at −80° C.

Expression Plates

-   -   1. Master Plates were thawed at room temperature or 30° C. prior        to replication.    -   2. Master Plates were replicated using a 384 well pintool to        inoculate the Expression Plates containing 60 μL of media. The        same media was used for the Expression Plates as the Master        Plates.    -   3. Expression Plates were grown overnight (approximately 16 hrs)        at 30° C. in a humidified incubator.    -   4. Expression Plates for the GSSM screen were induced with 200        ng/mL of anhydrous tetracycline (AHT) and the GeneReassembly        plates were induced a final concentration of 0.3 mM IPTG.    -   5. Expression Plates were grown overnight at 30° C. in a        humidified incubator.    -   6. Expression Plates were then stored at −20° C. until frozen,        usually overnight, to lyse the host cells.    -   7. Prior to assaying the Expression Plates they were thawed at        room temperature or 30° C.        Robotic Thermal Tolerance Screen    -   1. The robot was programmed to transfer 10 μL from the        Expression Plates to an Assay RT Plate and an Assay Heat Plate.    -   2. The Assay RT Plate remained at room temperature while the        Assay Heat Plate was incubated at elevated temperature for 1        hour. During heat treatment the Assay Heat Plates were covered        with a foam top. Temperatures for the heat treatment are listed        in the following Table 11:

TABLE 11 Temperatures for primary and secondary robotic screens.Temperature Treatment of Temperature Treatment of Screen GSSM VariantsGeneReassembly Variants Primary 50° C., 55° C. 65° C. Secondary 57° C.,60° C. 70° C.

-   -   3. After the heat treatment 40 μL of the substrate,        methylumbelliferyl myo-inositol phosphate (MUPI), was added        using a titertek. The substrate was prepared at a concentration        of 3 mM so that the final concentration in the Assay Plates is        2.5 mM.    -   4. The Assay Plates were incubated at room temperature for 5        min. The fluorescence was then measured as relative fluorescent        units (RFU) on a fluorescence reader at an excitation wavelength        of 360 nm and an emission of 465 nm.

Calculation of % Residual Activity

-   -   1. Each Assay plate contained 12 positive and 12 negative        controls. The positive controls contained the wild type enzyme        and the negative controls contained the vector only within the        host organism.    -   2. The fluorescence from the negative controls was averaged for        each 384 well plate and subtracted from the fluorescence of the        GSSM or GeneReassembly variants for the Assay RT and Assay Heat        plates.    -   3. Each well of the Assay Heat Plate was then divided by the        corresponding well from the Assay RT Plate to get a percent        residual activity (% RA) for each variant.    -   4. The % RA was used to rank the most thermal tolerant variants        from the high throughput robotic screen. These hits were        confirmed in additional assays

Secondary Screen

-   -   1. Improved thermal tolerance was confirmed using secondary        screens on selected hits. The hits were cherry picked from the        primary screen Master Plates into new Master Plates and assayed        at elevated temperatures listed in Table 11. The assay protocol        was the same as the protocol detailed above for the primary        screen.

TABLE 12 Summarizes the sequences and percent (%) residual activity ofthe top thermal tolerant GSSM variants selected for the construction ofthe GeneReassembly library. % Residual Activity AA Site Original AA NewAA 55° C. 57° C. 60° C. 105 D G 81.0% 58.8% 3.5% 175 N P 119.9% 75.7%2.1% 176 N F 136.7% 103.8%  10.1%  176 N L 102.2% 97.2% 9.8% 176 N W131.9% 61.7% 3.2% 176 N Y 180.0% 106.0%  4.1% 191 Q G 124.6% 77.6% 2.3%205 Y L 174.3% 114.2%  0.0% 244 N T 174.6% 95.7% 5.0% 252 Y L 148.4%62.0% 22.2%  252 Y R 149.3% 187.2%  15.4%  276 Y F 161.6% 112.1%  1.1%282 S C 161.6% 116.4%  8.9% 282 S H 256.8% 142.0%  0.7% 282 S L 143.6%97.8% 0.1% 282 S P 147.6% 96.8% 1.0% 282 S R 101.6% 72.5% 4.9% 284 L F85.9%   72%   0% 291 R N 86.4% 75.5% 5.6%

Point-mutants and associated data for Table 12 are shown immediatelybelow as Table 13. Note, in Table 13, the numbering of amino acidpositions begins with the added starting Methionine (e.g. amino acid “M”is position 1, amino acid “A” is position 2, amino acid “S” is position3, amino acid “S” is position 4, amino acid “I” is position 5, aminoacid “N” is position 6, and so on). This is important to make clear: forthe variants of SEQ ID NO:6, the numbering of the amino acid changesbegins with amino acid 31 of SEQ ID NO:6, were amino acid 31 (“A”) isreplaced with methionine (“M”).

TABLE 13 Amino Acid Original New Amino RT plate (data in relativePosition Amino Acid Acid 50° C. 55° C. 57° C. 60° C. fluorescence units,RFUs) 5 I R 109.5% −72.4% −27.7%  −83.3% 119213 10 N P 142.6% −97.8%−167.1%  −149.5%  47737 12 S C 98.6% 37.8% 19.6%  −1.6% 3926817 17 P R105.9% 70.7% 50.6%  0.6% 2834413 20 D R 168.8% 56.2%  9.5% −37.7% 24302422 I R 164.2% −56.3% −8.8% −67.9% 501992 30 P Q 69.1% 134.8% 119.4%  56.4% −98428 31 G L 59.7% 321.6% 52.3%  66.5% −89982 32 T R 112.1%234.3% 158.3%  141.4% −32840 32 T P −12.9% −39.0%  5.5%  56.1% 506296 32T N −41.5% 25.5% −6.8%  15.2% 215696 34 D G 111.4% 105.7% 112.3%   17.0%292094 34 D V 98.0% −39.1% 19.5%  −8.2% 623943 34 D S 108.0% 118.4%67.9% −18.3% 400778 48 W C 0.7%  −88%    48% 279831 52 Q G −19.0% −31.2%45.1% −11.0% 440078 52 Q L −24.5% −18.9% −17.5%  −15.0% 243731 52 Q R−13.8% −103.8% −45.8%  −357.5%  64939 56 F P −19.0% −41.0% −17.8% −14.6% 528700 57 R P 36.5%  −5%     6% 673072 57 R H 196.2% 54.8% 11.4% −2.7% 1766819 57 R W 94.5%  32%   −4% 888544 58 Y G 87.4%   8%     0%2203075 59 Q P 125.1% −1003%   211% 30707 64 A P −17.1%  −38%    21%298218 67 F A −84.1% −167%   −46% 132639 68 D G 859.6% 2454%   113%−5565 69 I R 550.5% 219.2% 283.9%  123.1% −39813 69 I S −26.7%   4%  −2% 837975 79 I R 111.4% 216.0% 308.4%  539.6% −47241 79 I C −9.4% 18%  −19% 901719 79 I S 242.7% −642%   −145% −33068 103 L E 391.0%−504%   492% 26863 103 L G 312.7% 333.5% 394.9%  317.6% −14442 103 L R135.6%  279%  238% −42440 103 L A 13.2%  −7%    38% 520801 103 L S105.0% 48.7% −162.4%  −90.0% 53552 103 L N −331.1% −381%   −229% 51581104 K P −1319.4% 1845%   4021% −7815 105 D G 106.9% 81.0% 58.8%  3.5%1739646 107 P H 30.7%   7%   −3% 2397754 107 P R 21.4%   4%   −3%3155286 107 P L 65.8% 55.2% 34.4%  −4.7% 447512 108 S G 95.5% 65.1%49.6%  −4.3% 307147 110 T F −10.5%  −7%     2% 875451 110 T K 126.1%52.7% 18.9% −49.3% 134884 112 I A −23.7%  −20%   −4% 453446 112 I K55.2% 95.2% 15.4% −46.1% 59053 115 L E −102.6%  −89%  103% 48860 115 L N−12.4%  −15%  −10% 1025328 115 L S −5.3% −8.9% −10.3%  −10.1% 556124 115L G −21.1% −36.4% −10.3%  −41.2% 644336 115 L R 106.0%  −28%  −215%−55337 116 K T 18.1%   7%    18% 230855 116 K V 45.1% −4.6% −19.7% −14.4% 235560 116 K L 78.8% −0.9% −37.9%  −19.2% 343119 116 K P 97.5%16.1% −20.9%  −32.3% 182525 116 K C −26.1%  −64%  −81% 226865 116 K F−560.3% −780%   −126% 49518 116 K Y −245.0% −488%   −141% 39189 117 K G10.2%  −26%  −21% 387561 117 K S −31.6%  −25%  −23% 548036 118 E K167.4%  409%  163% −44595 118 E Y 106.8%  −57%    40% 132171 118 E G45.4% −7.5% −11.7%  −10.8% 408480 118 E P 100.9% −77.5% −82.4%  −11.7%227778 118 E W −140.1%  −30%  −49% 115308 118 E A −61.4% −217%   −58%106520 118 E V −112.9%  57%  −89% 54418 118 E S −111.6% −294.0% −83.6% −172.0%  84511 118 E L −615.3% −772.3% −540.2%  −764.3%  6410 127 S G167.2%  24%   −2% 2332741 129 F S −103.1% −140.8% −135.6%  −138.6% 38893 129 F K 166.2%  46%  −224% −32568 130 S A 209.1%  13%   −3%2817649 133 F S −96.6%  245%  124% 68055 134 E G −11.5%  −6%   −6%1437969 134 E P −50.2% −179.6% −112.4%  −71.5% 107620 136 N P −7.0%  −9%  −7% 967546 139 R S 95.2%   9%     7% 3316801 139 R M 65.5%   3%   −2%3452962 139 R P 176.0%  53%   −3% 2170436 140 D T 7.5%  −4%     0%3067537 141 P L 12.2%  −3%   −1% 3026938 142 I P 105.3% 42.2% 15.3% −4.4% 1571795 142 I R −9.6%  −5%   −5% 2239061 142 I G −2.2%  −7%   −7%1821775 143 F G 84.3%  −10%    14% 547682 143 F V 7.9% −5.3% −3.4% −3.4% 2475003 143 F S 27.1% −10.3% −12.1%  −11.6% 492372 143 F T 7.0% −22%  −15% 1395343 144 L R −3.1%  −8%     3% 3104379 144 L P 7.8%  −2%    0% 1673141 151 K T 112.2%   6%     0% 3537421 153 G M 103.8%   1%  −7% 2086156 153 G V 97.6%  −8%  −13% 1534593 154 D R −4.6%  −3%   −3%1464648 155 A R 101.5% 149.6% 225.5%  159.3% −37404 155 A P 505.6%  86% 111% −84103 159 I T 80.3% 9.4% −3.2%  −4.9% 1414669 160 V R −72.5%  −8% −88% 360024 162 L S 77.9% −1.3% −0.7%  9.5% 653562 162 L F 126.7%  11%    5% 2305681 162 L G 84.3% 14.3% −14.4%  −24.4% 822742 162 L E −8.9%−25.7% −27.5%  −99.2% 255160 162 L D −252.6% −180%   −107% 41739 162 L R8.6% 10.3% −856.1%  −184.2%  −45546 163 K E −6.9%  −10%    10% 1257238163 K W −6.8%  −25%  −11% 935455 164 R L 684.4% 2692%   890% −11232 164R T −267.9% −259%   −276% 35611 165 Y E 4.2% −3.1% −3.5%  −3.1% 1330418165 Y S −0.6% −4.5% −5.0%  −4.5% 1204269 165 Y D −9.9%  −26%   −8%849532 165 Y G −21.0%  −9%  −12% 703657 174 Y R −27.9% −10.0% −12.8% −11.4% 478910 175 N P 186.8% 119.9% 75.7%  2.1% 3423684 176 N F 151.7%136.7% 103.8%   10.1% 3166873 176 N L 159.0% 102.2% 97.2%  9.8% 1973048176 N Y 215.1% 180.0% 106.0%   4.1% 2686812 176 N W 153.3% 131.9% 61.7% 3.2% 3375824 179 W V 654.7% −2109%   736% 21624 179 W L 211.2%  −87% −107% 66095 187 s V 81.7%  20%     9% 3668103 191 Q G 141.5% 124.6%77.6%  2.3% 4030462 193 V L 229.3% 177.3% 85.1%  −0.3% 2339956 196 T P253.8% 195.0% 122.7%  363.7% −81049 197 V R 84.1% 159.2% 43.9%  67.0%−67056 201 Y R 97.6%  244%  571% −94821 201 Y A 105.4%  168%  262%−79800 201 Y L 114.7% 108.5% 94.5% 111.0% −69345 201 Y P 99.5%  87% 109% −77061 201 Y Q 103.9% 100.2% 114.2%  100.9% −78259 201 Y E 98.4%196.7% 94.8%  22.0% −75564 201 Y S 93.7% 276.0% −294.6%  −67.2% −89008201 Y H −469.7% −450%   −233% 38056 205 Y L 176.2% 174.3% 114.2%   −0.4%6193145 206 D C 68.6%   7%     2% 2578407 208 K S −95.6%  −56%  −50%101991 215 T L 241.1% 100.4% 42.5%  −2.6% 2276174 216 L A 2.1%  −22%    9% 739181 222 N P −1.8%  −27%    16% 605972 238 S G 188.7% 120.1%64.7%  −8.5% 1960910 244 N T 147.2% 174.6% 95.7%  5.0% 3312364 244 N S144.6% 234.0% 148.4%   1.1% 2624829 252 Y L 131.0% 148.4% 62.0%  22.2%6033264 252 Y R 220.0% 149.3% 187.2%   15.4% 3753916 252 Y I 96.9% 41.4% 3.9% 10341043 261 M I 129.8% 117.8% 90.7%  −1.0% 3413811 268 R S −22.2% −19%  −23% 353376 268 R L −513.9% −333%   −149% 49591 272 I S 163.2%519.8% 300.9%  1188.1%  −42385 272 I R 4.9% 485.2% 277.9%  404.0% −76173272 I G 20.2%  161%  102% −85289 272 I E 199.6% 142.4% 163.1%   97.2%−65592 272 I N −91.2% −208%   −77% 99735 272 I P 92.7% −130.0% −142.8% −113.3%  73365 276 Y F 193.7% 161.6% 112.1%   1.1% 2536481 282 S C153.5% 161.6% 116.4%   8.9% 1804086 282 S R 108.4% 101.6% 72.5%  4.9%3108844 282 S P 158.8% 147.6% 96.8%  1.0% 3082327 282 S H 193.9% 256.8%142.0%   0.7% 1966076 282 S L 164.4% 143.6% 97.8%  0.1% 2208563 282 S E159.0% 199.7% 77.4%  −1.0% 2499129 282 S W 116.8%   6%   −3% 2625324 282S K 180.0% 181.2% 93.1%  −5.0% 1412699 282 S F 144.9% 113.1% 28.7% −5.1% 1668567 284 L F 85.9%  72%   −1% 3744515 287 Q L 106.5% 68.1%34.0%  −7.2% 2032181 291 R N 143.8% 86.4% 75.5%  5.6% 3463258 296 L E−169.4%  20%    31% 188612 Negative control 103.5%  130%  182% −60543Negative control 161.4% 136.6% 156.0%  157.1% −47900 Negative control450.3%  69%  156% −63840 Negative control 125.1%  267%  137% −67530Negative control 483.8% 140.8% 138.9%  135.3% −33533 Negative control508.5% 123.0% 138.5%  116.6% −44840 Negative control 104.3%  119%  114%−58308 Negative control 150.3% 96.9% 96.4%  89.8% −57605 Positivecontrol 106.8%  12%     6% 3745053 Positive control 122.1%   3%     6%2931573 Positive control 106.6%   8%     6% 3418796 Positive control92.3% 70.7% 55.1%  3.9% 5793014 Positive control 55.1%   7%     2%3312736 Positive control 153.4% 89.3% 51.6%  1.3% 4765881 Positivecontrol 132.9% 70.0% 34.0%  0.4% 5520616 Positive control 154.3% 80.7%17.6%  −0.7% 4018936

GeneReassembly was performed on nucleic acids as described herein usingthe top thermostable mutants from the GSSM phase; and assay conditionsfor the GeneReassembly variants described above in section entitled “SOPfor High Throughput Thermal Stability Assay”, of this example, above.(To reiterate: the nucleic acids encoding the thirty one (31) aminoacids of SEQ ID NO:6 (encoded e.g., by SEQ ID NO:5) were removed and anucleotides encoding a starting methionine were added for the nucleicacid that was “evolved” in the GSSM and GeneReassembly).

The best combination of enzyme variants after GeneReassembly (on thethermostable mutants from the GSSM phase) are set forth in Table 14,below. The invention provides enzymes, and the nucleic acids that encodethem, comprising any one, several or all of the amino acid changesdescribed in Table 14. For example, from the first row of Table 14, oneexemplary enzyme of the invention is an enzyme comprising an amino acidsequence as set forth in SEQ ID NO:6, but with amino acid changes asfollows: N176F, Q191G, Y205L, N244T, Y252R, Y276F, S282H, L284F and/orR291N.

Activity data for these exemplary enzymes of the invention is set forthin Table 15, below.

TABLE 14 best combination of enzyme variants after GeneReassembly AminoAcid Change ID N175 N176 Q191 Y205 N244 Y252 Y276 S282 L284 R291 A1 F GL T R F H F N B1 P Y Y178H G L T R F H N  3 P Y G L T R F H F N  4 P L GL T R F L F N  5 F G T R F H N  6 P Y G L R M261I F R N  8 P Y G L T R FR N B2 P F G L T L F H N 11 F G T R F H N 12 Y G L T F H F N 14 P F G LT R F L F N G2 F G L T R M261I F H N 17 P Y G L T R F L F N 18 F G T R FH F 20 F G L T R F H F N 21 L G L T L F H F 22 P Y G L T F H N 23 F G TR F R F 24 P Y G L T L F R F A4 F G L T L F R F 26 F G L T L F R 27 Y GL T R F H F N 28 P G L R F H F N 29 P F G L T L F H F N 30 P L L T R F PF 32 P W G L T R F R F 33 P Y L T R F H F N 34 P Y G T L F R F N 35 F GL T R F R F N 36 P W G L T L F R N 37 F G L T L F P 39 F G T L F H F N40 P Y G T R F R F N 41 F G L T F R F N 42 P Y G T R F R F N 43 P Y G LL H F N 44 P W G L T R F R F 45 P Y G T R F N 46 P W G L T L F R 47 P YG L L F R F N 48 P Y G L L H F 49 P G T R F H N 50 L G L T L F H 51 P FG L R H F N 52 P Y G T L F L F 53 Y G L N210N R F H F N 54 F G L R H N55 P W G L R F P F 57 P Y G L T R R F 58 P F G L T L F H 59 P F L T R FH N 60 P W G L T L F R F 61 P G L T L H F NData for the GeneReassembly variants is:

TABLE 15 65C 70C Residual Residual Additional Name Activity ActivityN175 N176 Q191 Y205 N244 Y252 Y276 S282 L284 R291 mutation A1 106.2437.14 F G L T R F H F N B1 111.5 23.4 P Y G L T R F H N Y178H  3 106.7725.48 P Y G L T R F H F N  4 105.4 15.89 P L G L T R F L F N  5 88.8416.39 F G T R F H N  6 100.71 20.78 P Y G L R F R N M261I  8 105.6416.94 P Y G L T R F R N B2 101.44 22.03 P F G L T L F H N 11 93.96 22.8F G T R F H N 12 95.32 18.59 Y G L T F H F N 14 83.24 22.83 P F G L T RF L F N G2 97.66 83.7 F G L T R F H N M261I 17 95.46 40.92 P Y G L T R FL F N 18 95.98 16.72 F G T R F H F 20 93.48 28.26 F G L T R F H F N 2193.52 15.54 L G L T L F H F 22 82.46 17.4 P Y G L T F H N 23 86.13 17.28F G T R F R F 24 121.7 44 P Y G L T L F R F A4 148.28 35.27 F G L T L FR F 26 108.03 18.92 F G L T L F R 27 85.02 16.09 Y G L T R F H F N 2886.9 16.52 P G L R F H F N 29 157.32 22.71 P F G L T L F H F N 30 86.4915.47 P L L T R F P F 32 112.42 18.88 P W G L T R F R F 33 121.27 15.74P Y L T R F H F N 34 88.4 30.67 P Y G T L F R F N 35 109.4 58.72 F G L TR F R F N 36 90.24 18.57 P W G L T L F R N 37 92.94 16 F G L T L F P 39104.51 19.07 F G T L F H F N 40 102.54 57.09 P Y G T R F R F N 41 94.2327.83 F G L T F R F N 42 112.91 16.34 P Y G T R F R F N 43 168.76 15.56P Y G L L H F N 44 254.08 28.87 P W G L T R F R F 45 141.67 15.51 P Y GT R F N 46 175.31 23.58 P W G L T L F R 47 172.6 35.64 P Y G L L F R F N48 109.25 15.12 P Y G L L H F 49 95.01 15.33 P G T R F H N 50 81.0116.01 L G L T L F H 51 109.05 15.06 P F G L R H F N 52 84.83 15.32 P Y GT L F L F 53 97.17 16.81 Y G L R F H F N N210N 54 127.34 15.05 F G L R HN 55 97.32 15.11 P W G L R F P F 57 149.08 19.49 P Y G L T R R F 58106.56 17.47 P F G L T L F H 59 89.33 15.06 P F L T R F H N 60 89.9417.85 P W G L T L F R F 61 108.91 17.96 P G L T L H F NLead GeneReassembly hit=“G2”, or SEQ ID NO:8 encoded by SEQ ID NO:7:

SEQ ID NO: 7: ATGGCTAGCTCTATTAATGTGCTTGAAAATTGGTCTAGATGGATGAAACCTATAAATGATGACATACCGTTAGCACGAATTTCAATTCCAGGAACACATGATAGTGGAACGTTCAAGTTGCAAAATCCGATAAAGCAAGTGTGGGGAATGACGCAAGAATATGATTTTCGTTATCAAATGGATCATGGAGCTAGAATTTTTGATATAAGAGGGCGTTTAACAGATGATAATACGATAGTTCTTCATCATGGGCCATTATATCTTTATGTAACACTGCACGAATTTATAAACGAAGCGAAACAATTTTTAAAAGATAATCCAAGTGAAACGATTATTATGTCTTTAAAAAAAGAGTATGAGGATATGAAAGGGGCGGAAAGCTCATTTAGTAGTACGTTTGAGAAAAATTATTTTCGTGATCCAATCTTTTTAAAAACAGAAGGAAATATAAAGCTTGGAGATGCTCGTGGGAAAATTGTATTACTAAAAAGATATAGTGGTAGTAATGAATCTGGGGGATATAATTTTTTCTATTGGCCAGACAATGAGACGTTTACCTCAACTATAAATGGTAATGTAAATGTAACAGTACAAGATAAATATAAAGTGAGTTTGGATGAGAAAATAAACGCTATTAAAGATACATTAAATGAAACGATTAACAATAGTGAAGATGTTAATCATCTATATATTAATTTTACAAGCTTGTCTTCTGGTGGTACAGCATGGACGAGTCCATATTATTATGCGTCCAGGATAAATCCTGAAATTGCAAATTATATTAAGCAAAAGAATCCTACGAGAGTGGGCTGGATAATACAAGATTTTATAAATGAAAAATGGCATCCATTACTTTATCAAGAAGTTATAAATGCGAATAAGTCACTTGTAAAATGA SEQ ID NO: 8:    MASSINVLENWSRWMKPINDDIPLARISIPGTHDSGTFKLQNPIKQVWGMTQEYDFRYQMDHGARIFDIRGRLTDDNTIVLHHGPLYLYVTLHEFINEAKQFLKDNPSETIIMSLKKEYEDMKGAESSFSSTFEKNYFRDPIFLKTEGNIKLGDARGKIVLLKRYSGSNESGGYNFFYWPDNETFTSTINGNVNVTVQDKYKVSLDEKINAIKDTLNETINNSEDVNHLYINFTSLSSGGTAWTSPYYYASRINPEIANYIKQKNPTRVGWIIQDFINEKWHPLLYQEVINANKSL VKOil Screen Data:

small scale screening for phospholipid content for GeneReassembly hitsis summarized in the table below. Oils were treated (or not treated, inthe case of the control) with enzyme as described in the protocolentitled “Small scale oil procedure”, below. Samples were then analyzedfor phospholipid content using NMR using the following protocol:

Small Scale Oil Procedure

Objective: To examine the activity of ePLC and PI-PLC in crude soybeanoil at timepoints during the enzyme reaction.

Oil:

Crude Soybean oil

FFA: 0.24%

pH: 6.97

DAG: 0.27% 1,2+0.24% 1,3=0.51% total DAG

PLs: 0.21% PA, 0.43% PE, 0.25% PI, 0.44% PC (1.34% total PLs); No LPA,0.01% LPE, No LPI, 0.02% LPC, No 1-LPLs; 0.01% A, No E, I or C; 661 ppmtotal phosphorus of which 628.6 ppm is from PLs

CP: 742 ppm P, 73.8 ppm Ca, 69.8 ppm Mg, 0.0 ppm Fe

Enzymes:

Evolved phosopholipase 8 (Example 2, Table 9)—11.5 Units/mg

Want 5.5 units×15 samples=82.5 units total/11.5 Units/mg ˜7 mg

Weighted out 12.1 mg×11.5 U/mg=139.15 Units resuspended in 120 uL 20 mMHepes pH 7.4, 1 mM ZnSO=1.16 units/uL

Want 5.5 units in 10 uL

5.5 units/(1.16 units/uL)=4.7 uL+5.3 uL=10 uL

Prepare stock 94 uL of 1.16 Units/uL and 106 uL 20 mM Hepes pH 7.4, 1 mMZnSO4.

Add 10 uL to reaction

SEQ ID NO:8-4.2 Units/mg

Want 0.02 units×15 samples=0.3 units total/4.2 Units/mg ˜1 mg

Weighted out 4.2 mg×4.2 U/mg=17.64 Units resuspended in 120 uL 20 mMHepes pH 7.4, 1 mM ZnSO4=0.147 units/uL

Want 0.02 units in 10 uL

0.02 units/(0.147 units/uL)=0.14 uL+9.86 uL=10 uL

Prepare stock 3 uL of 0.147 Units/uL and 197 uL 20 mM Hepes pH 7.4, 1 mMZnSO4.

Add 10 uL to reaction

Reaction Conditions:

1 mL of oil was aliquoted into 2 mL tubes using a Glison distriman.

Oil was preheated at 60 C shaking at 1400 rpm in thermomixer for ˜30minutes before addition of enzyme.

Enzyme was added to each sample then polytroned for 30 seconds andincubated at 60 C with continuous shaking.

Samples were removed at timepoints.

Immediately after removal of samples at timepoints, samples wereprepared for NMR analysis. The addition of NMR detergent pH 10.5 whichshould stop enzyme reaction.

Preparation of Reagents, Standards, and Samples for ³¹P NMRDetermination of Phospholipids and Products:

Two internal ³¹P standards of either TMP(2,2,6,6-Tetramethylpiperidine)/tributylphosphate (TBP) orTMP/trimethylpsoralen (TIP) at pH 10.5, were used. The TIP is mostimmune from spectral overlap but it does have a longer relaxation time(2.76 sec) compared with 1.02 sec for TBP. TBP matches the T₁ values ofPC, PE and PI whereas TIP matches more with PA. Saturation factors havebeen calculated from data obtained with normal recycle delay of 1.74 secvs. a 21.6 second recycle using a 58 degree tip angle for optimum S/Nper unit time using TIP. This is probably the preferred method. TBPthough more efficient because of the shorter T₁ has a chemical shiftintermediate between PI and PC and is highly temperature dependent andsuffers from overlap.

This provides the following advantages:

-   -   (i) pH 10.5 cleanly separates LPI from PE (they are overlapped        at pH 8.6, and 9.5),    -   (ii) TBP and TIP internal standards allows more rapid recycling        NMR delays with approximately 2.8 improvement in S/N per unit        time,    -   (iii) Provides an internal check of both 2 mM TBP/TIP and 2 mM        TMP references,    -   (iv) Allows different NMR conditions to be selected based on        needs (PL's or products, for example).

Preparation of Reagents

-   -   1. 5% Deoxycholic acid (DOC): dissolve 5.0 g of Deoxycholic acid        Na salt into 100 ml of HPLC grade water.    -   2. 50 mM EDTA/112.5 mM TRIS: add 1.46 g of EDTA acid and 1.3624        g of TRIS base to 100 ml of HPLC grade water.    -   3. 5:4 DOC/(EDTA/TRIS), pH 10.5 Detergent: mix 50 ml of DOC Na        and 40 ml of EDTA/TRIS. Add pellet wise KOH until pH is 10.5 (a        few pellets). This detergent contains 50 mM TRIS for pH        buffering.    -   4. In summary, to make 900 ml detergent: 25 g DOC, 5.84 g EDTA,        5.45 g Tris base, 900 ml H₂O, adjust the pH to 10.5 using KOH        pellets.    -   5. 50 mM TMP and 50 mM TBP Internal Standard in HPLC grade        isopropanol (IPA): first prepare 100 mM TMP (MW 140.08) and 100        mM TBP (MW 266.32) in IPA respectively, and then mix them at the        ratio of 1:1. Prepare a fresh stock each week of analysis and        store it at 4° C. to maintain stability.

Preparation of Standards and Samples

-   -   6. PL Calibration solution: accurately weigh (+/−0.1 mg)        approximately 10 mg of Avanti lecithin (PA 5.9%, PE 10.4%, PI        8.3%, PC 14.0%, LPC 0.5%) into a 2 ml vial. Add 40 μl of 50 mM        TMP/50 mM TBP internal standard, 100 μl D₂O, and 860 μl        Detergent and mix thoroughly by vortexing for half an hour, and        take 500 μl clear aqueous solution into a standard 5 mm NMR tube        after spinning for a while*. The concentration of TMP and TBP        are 2000 μM and 2000 μM respectively; the molecular weights of        PA, PE, PI, PC, and PLC are approximately 697, 716, 857, 758,        and 496 respectively, so for 10.0 mg/ml Avanti lecithin, PA is        0.846 mM, PE is 1.453 mM, PI is 0.968 mM, PC is 1.847 mM, and        LPC 0.101 mM.    -   7. Crude soy oil sample solution: vortex the oil and accurately        weigh (+/−0.2 mg) approximately 100 mg oil into a 2 ml vial. Add        100 ul D₂O, and 900 μl Detergent and mix thoroughly by vortexing        for half an hour. After spinning for a while, take 600 μl clear        aqueous solution into a vial* and add 24 μl of 50 mM TMP/50 mM        TBP internal standard and mix thoroughly, then take 500 μl clear        aqueous solution into a standard 5 mm NMR tube.    -   8. De-gummed oil sample solution: vortex the oil and accurately        weigh (+/−0.2 mg) approximately 250 mg oil into a 2 ml vial. Add        100 μl D₂O, and 900 μl detergent and mix thoroughly by vortexing        for half an hour. After spinning for a while, take 600 μl clear        aqueous solution into a vial* and add 24 μl of 50 mM TMP/50 mM        TBP internal standard and mix thoroughly, then take 500 μl clear        aqueous solution into a standard 5 mm NMR tube.    -   9. Gum sample solution: weigh approximately 10 mg gum (+/−0.1        mg) (no more than 11 mg) into a 2 ml vial. Add 40 ul of 50 mM        TMP/12.5 mM TBP internal standard, 100 ul D₂O, and 860 ul        Detergent and mix thoroughly by vortexing for half an hour, and        take 500 ul clear aqueous solution into a standard 5 mm NMR tube        after spinning for a while*. *The sample solution becomes two        layers after spinning A needle syringe is used to transfer the        lower layer of the clear aqueous solution into the NMR tube. Use        caution not to disturb the top layer.    -   10. Crude canola oil sample solution: vortex the oil and        accurately weigh (+/−0.2 mg) approximately 250 mg oil into a 2        ml vial. Add 100 ul D₂O, and 900 μl Detergent and mix thoroughly        by vortexing for half an hour. After spinning for a while, take        600 μl clear aqueous solution into a vial* and add 24 ul of 50        mM TMP/50 mM TBP internal standard and mix thoroughly, then take        500 μl clear aqueous solution into a standard 5 mm NMR tube.    -   11. Water Wastes sample solution: give an estimation of the % v        of the oil in the water waste. Vortex the water waste and take        0.5 ml into a 2 ml vial and accurately weigh it (approximately        500 mg). Add 100 μl D₂O and approximately 400 μl of detergent to        make a 1 ml solution (excluding the oil entrained in the water        wastes) and mix thoroughly by vortexing. After spinning for a        while, take 600 ul clear aqueous solution into a vial* and add        24 μl of 50 mM TMP/50 mM TBP internal standard and mix        thoroughly, then take 500 μl clear aqueous solution into a        standard 5 mm NMR tube. *The sample solution becomes two layers        after spinning A needle syringe is used to transfer the lower        layer of the clear aqueous solution into the NMR tube. Use        caution not to disturb the top layer.        Data Collection for ³¹P NMR Determination of Phospholipids and        Products

Data parameter sets have been set up for automated ICONNMR™ (BrukerBioSpin Corporation, Fremont, Calif.) operation with a 58 degree tipangle coded for the default high power. Insert sample into probe with“ej”/“ij” commands. Check edte=300.0. In TopSpin use the “rpar”operation first and read in the parameter set P31_TBP_TMP_std. Go to theacquisition window with “acqu” and tune the QNP probe for both 31P and1H using the “wobb” command. “ej” sample and then proceed with IconNMRautomation using the same parameter table. In automation samples arequeued and run in turn with shimming beforehand. Editable parameters areNS and D1. For allocated time NS=512-2048 provide adequate S/N. Scalingfactors to account for the different relaxation times have beenaccumulated and should be checked on an Avanti lecithin sample run withany samples.

TABLE 16 Oil screen data (small scale, phospholipid content) forGeneReassembly hits: Weight of oil Total Oil Enzyme Treatment Oil: Mix(mg) PA(%) PE(%) PI(%) PC(%) PL(%) LPA(%) Crude Soy Oil PI-PLC A1 200mgs 210.8 0.24 0.42 0.07 0.40 1.13 0.00 Crude Soy Oil PI-PLC A1 200 mgs214.7 0.23 0.42 0.06 0.39 1.09 0.00 Crude Soy Oil PI-PLC B1 200 mgs211.4 0.24 0.47 0.05 0.44 1.20 0.00 Crude Soy Oil PI-PLC B1 200 mgs214.5 0.24 0.44 0.18 0.43 1.28 0.00 Crude Soy Oil PI-PLC B2 200 mgs210.9 0.26 0.48 0.07 0.47 1.28 0.00 Crude Soy Oil PI-PLC B2 200 mgs211.4 0.26 0.48 0.06 0.47 1.26 0.00 Crude Soy Oil PI-PLC G2 200 mgs211.6 0.25 0.47 0.05 0.45 1.22 0.00 Crude Soy Oil PI-PLC G2 200 mgs 2100.24 0.45 0.05 0.43 1.18 0.00 Crude Soy Oil PI-PLC A4 200 mgs 209.9 0.240.43 0.15 0.41 1.23 0.01 Crude Soy Oil PI-PLC A4 200 mgs 208.5 0.25 0.460.10 0.43 1.24 0.00 Crude Soy Oil PI-PLC WT 200 mgs 212 0.22 0.41 0.200.41 1.24 0.00 (SEQ ID NO: 6) Crude Soy Oil PI-PLC WT 200 mgs 209.4 0.240.46 0.16 0.43 1.29 0.02 (SEQ ID NO: 6) Crude Soy Oil No Enzyme 200 mgs211.3 0.21 0.39 0.22 0.38 1.20 0.00 Crude Soy Oil No Enzyme 200 mgs209.9 0.26 0.44 0.26 0.41 1.37 0.00 Crude Canola Oil No Enzyme 200 mgs194.7 0.17 0.13 0.20 0.34 0.85 0.00 Crude Canola Oil No Enzyme 200 mgs194.9 0.17 0.14 0.21 0.34 0.85 0.00 Crude Canola Oil No Enzyme 200 mgs196.1 0.18 0.14 0.22 0.35 0.89 0.00

TABLE 17 Total P 1- 1- 1- 1- Total P from PLs Oil LPE(%) LPI(%) LPC(%)LPA(%) LPE(%) LPI(%) LPC(%) A(%) E(%) I(%) C(%) (ppm) (ppm) Crude SoyOil 0.01 0.00 0.02 0.00 0.00 0.01 0.00 0.01 0.00 0.02 0.00 644 478 CrudeSoy Oil 0.01 0.00 0.02 0.00 0.00 0.01 0.00 0.01 0.00 0.02 0.00 646 462Crude Soy Oil 0.01 0.00 0.02 0.00 0.00 0.02 0.00 0.01 0.00 0.02 0.00 725510 Crude Soy Oil 0.01 0.00 0.02 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00640 535 Crude Soy Oil 0.01 0.00 0.02 0.00 0.00 0.00 0.00 0.01 0.00 0.030.00 729 540 Crude Soy Oil 0.01 0.00 0.02 0.00 0.00 0.02 0.00 0.01 0.000.03 0.00 755 534 Crude Soy Oil 0.02 0.00 0.02 0.00 0.00 0.03 0.00 0.010.00 0.03 0.00 729 516 Crude Soy Oil 0.01 0.00 0.03 0.00 0.00 0.01 0.000.01 0.00 0.03 0.00 733 498 Crude Soy Oil 0.02 0.01 0.03 0.00 0.00 0.020.00 0.01 0.00 0.02 0.00 701 515 Crude Soy Oil 0.01 0.00 0.02 0.00 0.000.02 0.00 0.01 0.00 0.03 0.00 700 522 Crude Soy Oil 0.01 0.01 0.02 0.000.00 0.02 0.00 0.01 0.00 0.00 0.01 657 518 Crude Soy Oil 0.01 0.00 0.020.00 0.00 0.03 0.00 0.01 0.00 0.00 0.00 737 540 Crude Soy Oil 0.01 0.020.02 0.00 0.00 0.02 0.00 0.01 0.00 0.00 0.00 608 499 Crude Soy Oil 0.010.01 0.02 0.00 0.00 0.02 0.00 0.01 0.01 0.00 0.02 759 569 Crude CanolaOil 0.02 0.02 0.04 0.00 0.00 0.02 0.00 0.02 0.00 0.00 0.00 480 348 CrudeCanola Oil 0.01 0.01 0.04 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.00 474349 Crude Canola Oil 0.01 0.01 0.04 0.00 0.00 0.01 0.00 0.01 0.00 0.000.00 484 365

TABLE 18 AVERAGED DATA Total Oil Enzyme Treatment PA(%) PE(%) PI(%)PC(%) PL(%) LPA(%) LPE(%) LPI(%) LPC(%) Crude Soy Oil PI-PLC A1 0.240.42 0.06 0.39 1.11 0.00 0.01 0.00 0.02 Crude Soy Oil PI-PLC B1 0.240.45 0.12 0.43 1.24 0.00 0.01 0.00 0.02 Crude Soy Oil PI-PLC B2 0.260.48 0.06 0.47 1.27 0.00 0.01 0.00 0.02 Crude Soy Oil PI-PLC G2 0.240.46 0.05 0.44 1.20 0.00 0.01 0.00 0.03 Crude Soy Oil PI-PLC A4 0.240.45 0.13 0.42 1.24 0.01 0.01 0.00 0.02 Crude Soy Oil PI-PLC WT 0.230.44 0.18 0.42 1.27 0.01 0.01 0.01 0.02 (SEQ ID NO: 6) Crude Soy Oil NoEnzyme 0.23 0.42 0.24 0.40 1.29 0.00 0.01 0.01 0.02 Crude Canola Oil NoEnzyme 0.17 0.14 0.21 0.34 0.87 0.00 0.01 0.01 0.04

TABLE 19 AVERAGED DATA 1- 1- 1- 1- Total P Total P from Oil LPA(%)LPE(%) LPI(%) LPC(%) A(%) E(%) I(%) C(%) (ppm) PLs (ppm) Crude Soy Oil0.00 0.00 0.01 0.00 0.01 0.00 0.02 0.00 645.40 470.17 Crude Soy Oil 0.000.00 0.01 0.00 0.01 0.00 0.01 0.00 682.44 522.47 Crude Soy Oil 0.00 0.000.01 0.00 0.01 0.00 0.03 0.00 741.94 536.72 Crude Soy Oil 0.00 0.00 0.020.00 0.01 0.00 0.03 0.00 730.76 506.92 Crude Soy Oil 0.00 0.00 0.02 0.000.01 0.00 0.02 0.00 700.48 518.82 Crude Soy Oil 0.00 0.00 0.02 0.00 0.010.00 0.00 0.00 697.17 528.75 Crude Soy Oil 0.00 0.00 0.02 0.00 0.01 0.000.00 0.01 683.32 533.87 Crude Canola Oil 0.00 0.00 0.01 0.00 0.02 0.000.00 0.00 479.51 354.10

Large Scale Degumming

with PI-PLC and ePLC or PI-PLC with PLC (SEQ ID NO: 2), with the resultssummarized in the Tables 20, 21 and 22, below. Oils were treated (or nottreated, in the case of the control) with enzyme using this “Large ScaleOil Procedure”:

Large Scale Oil Procedure

Objective:

To compare the activity of PLC, PI-PLC, “Evolved” PLCs (or “ePLCs”) ofExample 2, above, in 2 k g of crude soybean oil.

Oil:

Crude Soybean oil

FFA: 0.24%

pH: 6.97

DAG: 0.27% 1,2+0.24% 1,3=0.51% total DAG

PLs: 0.21% PA, 0.43% PE, 0.25% PI, 0.44% PC (1.34% total PLs); No LPA,0.01% LPE, No LPI, 0.02% LPC, No 1-LPLs; 0.01% A, No E, I or C; 661 ppmtotal phosphorus of which 628.6 ppm is from PLs

CP: 742 ppm P, 73.8 ppm Ca, 69.8 ppm Mg, 0.0 ppm Fe

Enzymes:

PLC (SEQ ID NO:2)

Want 5.5 units=200 ppm=0.4 g/2000 g oil

Preheat oil to 60° C. with continuous mixing 200 rpm.

Move preheated oil to high shear mixer.

Start mixing at low speed.

Add 0.4 g PLC (SEQ ID NO:2)+60 g room temperature water to preheated oilwhile mixing.

Adjust high shear mixer to 6 (highest speed) for 1 minute.

Move sample back to paddlemixer and continuously stir at 200 rpm at 60°C. for 2 hours.

Adjust temperature to 80° C. Collect noncentrifuged oil sample and storeat RT for analysis

Once temperature of oil reaches 80 C, centrifuge using Gyro testercentrifuge.

Evolved phosopholipase 8 (Example 2, Table 9)—11.5 U/mg

Want 5.5 units/g oil, reaction 2 kg or 2000 g=11 000 units total

Want 11,000 units/11.5 U/mg=957 mg

Crude Oil:

Weighted out 958.6 mg×11.5 U/mg=11,024 Units. Resuspended samples in 60g water immediately before addition to crude oil.

Evolved phosopholipase 156 (Example 2, Table 9)—17.2 U/mg

Want 5.5 units/g oil, reaction 2 kg or 2000 g=11 000 units total

Want 11,000 units/17.2 U/mg=640 mg

Crude Oil:

Weighted out 642.49 mg×17.2 U/mg=11,049 Units. Resuspended samples in 60g water immediately before addition to crude oil.

SEQ ID NO:8-4.2 U/mg

Want 0.02 units/g oil, reaction 2 kg or 2000 g=40 units total

Want 40 units/4.2 U/mg=9.5 mg

Crude Oil:

Weighted out 9.6 mg×4.2 U/mg=40.32 Units. Resuspended samples in 60 gwater immediately before addition to crude oil.

Evolved phosopholipase 8+SEQ ID NO:8-2 hrs: Weighted out 959.2 mgEvolved phosopholipase 8 PH045×11.5 U/mg=11,031 Units. Weighted out 9.8mg SEQ ID NO:8×4.2 U/mg=41.16 Units

Evolved phosopholipase 8+SEQ ID NO:8-4 hrs: Weighted out 959.1 mgEvolved phosopholipase 8 PH045×11.5 U/mg=11,030 Units. Weighted out 9.8mg SEQ ID NO:8×4.2 U/mg=41.16 Units

Resuspended samples in 60 g water immediately before addition to crudeoil and Evolved phosopholipase 8+SEQ ID NO:8 at same time.

Reaction Conditions:

2000 g of oil was weighted into a 4 L beaker. The oil was preheated to60 C on hotplate with feedback temperature control (Barnstead/Themolynemirak)

Oil was preheated at 60 C with continuous stirring at ˜200 rpm beforeaddition of enzyme.

Samples were moved to high speed mixer, enzyme+60 g room temperaturewater to preheated oil while mixing at low speed then immediately highshear mixed (max speed) for 1 minute.

Move sample back to paddlemixer and continuously stir at 200 rpm at 60 Cfor 2 hours.

Adjust temperature to 80 C. Collect noncentrifuged oil sample and storeat −20 C for analysis

Once temperature of oil reaches 80 C, centrifuge using high speedcentrifuge.

TABLE 20 % Average Average Theoretical Free % 1,2- % 1,3- Sum 1,3 & Netmax DAG Fatty Acid Description of oil assay DAG DAG 1,2 DAG DAG obtained(titration) crude soybean oil 0.36 0.32 0.68 0.00 0.47 SEQ ID NO: 2treated 1.11 0.33 1.44 0.76 80 0.47 precentrifuged oil * SEQ ID NO: 2treated 0.98 0.33 1.32 0.64 68 0.24 centrifuged oil * Evolvedphosopholipase 8 1.56 0.33 1.88 1.20 128 0.48 treated precentrifugedoil * Evolved phosopholipase 8 1.78 0.39 2.16 1.48 158 0.24 treatedcentrifuged oil * Evolved phosopholipase 156 1.44 0.32 1.76 1.08 1140.49 treated precentrifuged oil * Evolved phosopholipase 156 1.48 0.341.82 1.14 121 0.25 treated centrifuged oil * SEQ ID NO: 8 treated 0.660.32 0.98 0.30 157 0.48 precentrifuged oil ** SEQ ID NO: 8 treated 0.550.34 0.90 0.22 112 0.21 centrifuged oil ** Evolved phosopholipase 8 +1.71 0.33 2.04 1.36 119 0.71 SEQ ID NO: 8 treated 2 hrs precentrifugedoil *** Evolved phosopholipase 8 + 1.87 0.37 2.23 1.55 136 0.24 SEQ IDNO: 8 treated 2 hrs centrifuged oil *** Evolved phosopholipase 8 + 1.990.38 2.37 1.69 148 0.72 SEQ ID NO: 8 treated 4 hrs precentrifuged oil*** Evolved phosopholipase 8 + 1.92 0.38 2.30 1.62 142 0.21 SEQ ID NO: 8treated 4 hrs centrifuged oil *** Net DAG = Sum 1,3 & 1,2 DAG Generated− Endogenous DAG % Theoretical max DAG obtained = (Net DAG/TheoreticalMax DAG)*100 crude soybean oil 06 17 08: PC = 0.47, PE = 0.46, PA =0.22, PI = 0.27 * Theoretical max DAG from NMR PL PC, PE & PA values =(% PC*0.78) + (% PE*0.83) + (% PA*0.89) = 0.94 ** Theoretical max DAGfrom NMR PL PI value = (% PI*0.72) = 0.194 *** Theoretical max DAG fromNMR PL PC, PE, PA & PI values = (% PC*0.78) + (% PE*0.83) + (%PA*0.89) + (% PI*0.72) = 1.14

TABLE 21 PA(%) PI(%) PE(%) PC(%) % PA Description of oil assays PA(%)PI(%) PE(%) PC(%) SD SD SD SD Removal crude soybean oil 0.21 0.26 0.430.45 0.01 0.01 0.01 0.02 0 SEQ ID NO: 2 treated 0.20 0.25 0.18 0.06 0.000.00 0.00 0.01 7 precentrifuged oil SEQ ID NO: 2 treated 0.05 0.00 0.000.00 0.00 0.01 0.00 0.00 77 centrifuged oil Evolved phosopholipase 80.04 0.22 0.00 0.00 0.00 0.01 0.00 0.00 80 treated precentrifuged oilEvolved phosopholipase 8 0.03 0.02 0.00 0.00 0.00 0.00 0.00 0.00 85treated centrifuged oil Evolved phosopholipase 156 0.05 0.08 0.01 0.000.01 0.01 0.01 0.00 75 treated precentrifuged oil Evolved phosopholipase156 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 83 treated centrifuged oilSEQ ID NO: 8 treated 0.20 0.00 0.42 0.41 0.01 0.00 0.01 0.00 0precentrifuged oil SEQ ID NO: 8 treated 0.07 0.00 0.05 0.02 0.01 0.000.01 0.00 65 centrifuged oil Evolved phosopholipase 8 + 0.07 0.00 0.030.00 0.01 0.00 0.01 0.00 67 SEQ ID NO: 8 treated 2 hrs precentrifugedoil Evolved phosopholipase 8 + 0.04 0.00 0.00 0.00 0.00 0.00 0.00 0.0082 SEQ ID NO: 8 treated 2 hrs centrifuged oil Evolved phosopholipase 8 +0.05 0.00 0.01 0.00 0.00 0.00 0.00 0.00 74 SEQ ID NO: 8 treated 4 hrsprecentrifuged oil Evolved phosopholipase 8 + 0.03 0.00 0.00 0.00 0.000.00 0.00 0.00 86 SEQ ID NO: 8 treated 4 hrs centrifuged oil

TABLE 22 PA(%) PI(%) PE(%) PC(%) Description of oil assays PA(%) PI(%)PE(%) PC(%) SD SD SD SD A(%) I(%) E(%) C(%) SEQ ID NO: 2 treated gums3.60 5.21 5.19 2.96 0.10 0.26 0.28 0.14 0.14 0.00 1.17 2.54 Evolvedphosopholipase 8 0.49 6.28 0.04 0.00 0.06 0.40 0.07 0.00 1.22 0.00 3.254.41 treated gums Evolved phosopholipase 156 1.32 6.67 0.56 0.00 0.060.24 0.06 0.00 0.84 0.00 2.85 4.02 treated gums SEQ ID NO: 8 treatedgums 2.65 0.63 7.20 7.15 0.10 0.04 0.34 0.24 0.09 0.86 0.03 0.12 Evolvedphosopholipase 8 + 1.35 0.90 0.85 0.28 0.06 0.13 0.05 0.26 1.23 2.983.50 4.90 SEQ ID NO: 8 treated 2 hrs gums Evolved phosopholipase 8 +0.95 0.31 0.39 0.08 0.11 0.06 0.03 0.13 1.34 4.50 3.57 4.83 SEQ ID NO: 8treated 4 hrs gums

TABLE 23 1- 1- 1- 1- X(uM) Description of oil assays LPA(%) LPE(%)LPI(%) LPC(%) LPA(%) LPE(%) LPI(%) LPC(%) 12.9 ppm crude soybean oil0.00 0.01 0.01 0.03 0.00 0.00 0.00 0.00 0 SEQ ID NO: 2 treated gums 0.230.40 0.58 0.46 0.05 0.00 0.10 0.00 0 Evolved phosopholipase 8 0.33 0.000.29 0.28 0.55 0.00 0.00 0.00 60.9 treated gums Evolved phosopholipase156 0.00 0.03 0.36 0.55 0.46 0.00 0.00 0.00 0 treated gums SEQ ID NO: 8treated gums 0.00 0.28 0.00 0.54 0.00 0.00 0.00 0.00 439.5 Evolvedphosopholipase 8 + 0.26 0.20 0.00 0.62 0.60 0.00 0.00 0.00 526.9 SEQ IDNO: 8 treated 2 hrs gums Evolved phosopholipase 8 + 0.16 0.07 0.00 0.340.63 0.00 0.00 0.00 265.5 SEQ ID NO: 8 treated 4 hrs gums

-   -   Samples were then analyzed for phopsholipid content (PL data)        using NMR, as described above. Samples were also analyzed for        DAG content (DAG FFA) using the following HPLC protocols:        Determination of Diacylglycerol in Vegetable Oil by High        Performance Liquid Chromatography with Evaporative Light        Scattering Detector

This method is based on AOCS method Cd 11d-96, as described in Mono- andDiglycerides Determination by HPLC-ELSD (AOCS Official Method Cd11d-96), with some modifications. One significant change is the adoptionof ENOVA™ oil as the standard for quantification purpose. The AOCSmethod uses dipalmitin (C16:0) as standard. However, in vegetable oil,C16:0 only accounts for ˜10%, while C18:0, C18:1, and C18:2 stand fornearly 90%. In HPLC chromatogram, not only is the peak shape ofdipalmitin different from that of the actual diacylglycerols (DAG) inthe vegetable oil, the detector's response to dipalmitin is alsodifferent from C18 DAG. Both factors affect the quantification resultbecause evaporative light scattering detector (ELSD) is a non-lineardetector. ENOVA™ oil is high-DAG oil produced through a patented processby ADM using soybean oil and canola oil as raw material, which has afatty acids distribution similar to regular vegetable oil and hence abetter standard for quantification of the DAG in vegetable oil. Theamount of DAG in ENOVA™ oil can be determined using AOCS Official MethodCd 11b-91 (2) and ³¹P NMR method (3, 4).

Preparation of Sample and Standard Solutions:

-   1. Sample solution: accurately weight approximately 50 μl oil    samples and add 950 ul hexane/isopropanol=9:1 to make 1 ml solution.-   2. Standard solutions: the range of 1,2-DAG and 1,3-DAG in standard    solutions shall cover the actual DAG concentration in sample    solution. One example is 5 ENOVA™ oil solutions with concentration    of 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, and 4 mg/ml    respectively.

HPLC Settings:

Column: Chromegasphere SI-60, 15 cm×4.6 mm

Temperature: 40° C.

Flow Rate: 2 mL/min

Injection volume: 20 ul

Mobile phase A: Hexane

Mobile phase B: Hexane/Isopropanol/Ethyl Acetate/Formicacid=800:100:100:1

Gradient Elution:

Time (min) 0 8 8.5 15 15.1 19 % B 2 35 98 98 2 2

ELSD Settings:

The parameters of Sedex 75 ELSD shall be optimized to maximize thesensitivity. These include temperature, gain, nebulizer gas pressure,and the position of glass cell. A typical example is temperature 40° C.,gain 5, and nitrogen gas 3.5 bars.

Peak Identification and Quantification

Identify DAG peak by comparison of retention time with that of standard.Quantification is based on the relationship between the detector'sresponse I (Peak Area) and the analyte's concentration C: I=K*C^(M),here K and M are experimental conditions related constants.

REFERENCES

-   1. Mono- and Diglycerides Determination by HPLC-ELSD (AOCS Official    Method Cd 11d-96).-   2. Determination of Mono- and Diglycerides by Capillary Gas    Chromatography (AOCS Official Method Cd 11b-91).-   3. Spyros, A.; Dais, P. Application of ³¹P NMR Spectroscopy in Food    Analysis. 1. Quantitative Determination of the Mono- and Diglyceride    Composition of Olive Oils, J. Agric. Food Chem. 2000, 48, 802-805.-   4. Vigli, G.; Philippids, A.; Spyros, A.; Dais, P. Classification of    Edible Oils by Employing ³¹P and ¹H NMR Spectroscopy in Combination    with Multivariate Statistical Analysis. A Proposal for the Detection    of Seed Oil Adulteration in Virgin Olive Oils, J. Agric. Food Chem.    2003, 51, 5715-5722.    Lead GR Hit “G2” was Codon Optimized:

Codon optimization was attempted two different ways on the subcloned ORF(G2 codon opt V1 and G2 codon opt V2) and with the second methodproviding a much more highly expressable variant. For G2 codon opt V1,original codons whose in the subcloned ORF usage fell below 15-25% forP. fluorescens were changed to codons in P. fluorescens with usage>30%.Additionally two TGA stop codons were used at the 3′ end of the ORF.

G2 codon opt V1 (SEQ ID NO: 9):ATGGCCAGCAGCATCAACGTCCTCGAAAACTGGTCGCGCTGGATGAAGCCGATCAACGACGACATCCCGCTGGCCCGCATCAGCATCCCGGGCACCCACGACAGCGGCACCTTCAAGCTGCAGAACCCGATCAAGCAGGTCTGGGGCATGACCCAGGAATACGACTTCCGCTACCAGATGGACCACGGCGCCCGCATCTTCGACATCCGCGGCCGCCTGACCGACGACAACACCATCGTGCTGCACCACGGCCCGCTGTACCTGTACGTGACCCTGCACGAATTCATCAACGAAGCCAAGCAGTTCCTGAAGGACAACCCGAGCGAAACCATCATCATGAGCCTGAAGAAAGAATACGAAGACATGAAGGGCGCCGAAAGCAGCTTCAGCAGCACCTTCGAAAAGAACTACTTCCGCGACCCGATCTTCCTGAAGACCGAAGGCAACATCAAGCTGGGCGACGCCCGCGGCAAGATCGTCCTCCTGAAGCGCTACAGCGGCAGCAACGAAAGCGGCGGCTACAACTTCTTCTACTGGCCGGACAACGAAACCTTCACCAGCACCATCAACGGCAACGTGAACGTGACCGTGCAGGACAAGTACAAGGTGAGCCTGGACGAAAAGATCAACGCCATCAAGGACACCCTGAACGAAACCATCAACAACAGCGAAGACGTGAACCACCTGTACATCAACTTCACCAGCCTGAGCAGCGGCGGCACCGCCTGGACCAGCCCGTACTACTACGCCAGCCGCATCAACCCGGAAATCGCCAACTACATCAAGCAGAAGAACCCGACCCGCGTGGGCTGGATCATCCAGGACTTCATCAACGAAAAGTGGCACCCGCTGCTGTACCAGGAAGTGATCAACGCGAACAAGAGCCTGGTCAAGTGATGA

For G2 codon opt V2, P. fluorescens codons were chosen so that theirusage (%) most closely matched the usage (%) of each codon as found inthe original ORF. This is a process we call Codon Usage Transfer.Additionally the predicted mRNA secondary structure of the transcriptwas minimized to prevent stem loops and hairpins at the translationstart (a window of nucleotides in the mRNA from −65 to +65, wheretranslation starts at +1 with the “A” nucleotide of “ATG” which is codesfor the first methionine of the protein). In this minimization,synonymous codons with similar usage (%) were used to reduce unwantednucleotide-nucleotide pairings. As before, two TGA stop codons were usedat the 3′ end of the ORF.

G2 codon opt V2 (SEQ ID NO: 10):    ATGGCGAGCAGCATCAACGTCTTGGAGAACTGGTCCCGGTGGATGAAGCCCATCAACGACGATATCCCACTGGCCCGTATCTCGATCCCGGGCACCCACGACAGCGGCACCTTTAAACTCCAGAACCCAATCAAACAGGTCTGGGGCATGACCCAGGAGTACGACTTCCGCTACCAGATGGACCACGGCGCCCGGATCTTCGACATCCGGGGGCGCCTGACCGACGACAACACCATCGTGCTGCACCACGGGCCGCTGTACCTGTACGTGACCTTGCATGAGTTCATCAATGAGGCGAAGCAGTTCCTGAAGGACAACCCGAGCGAAACCATCATCATGTCCCTGAAGAAAGAATACGAAGACATGAAGGGGGCGGAGAGTTCGTTCAGCAGCACCTTCGAAAAGAACTACTTCCGCGACCCGATTTTCCTGAAGACCGAGGGCAACATCAAACTGGGCGACGCCCGCGGCAAGATCGTGCTGTTGAAGCGGTACAGCGGCAGCAACGAGTCCGGGGGCTACAACTTCTTTTACTGGCCGGATAACGAAACCTTCACTTCGACGATCAACGGCAACGTGAACGTGACCGTGCAGGACAAGTACAAGGTCAGCCTCGACGAAAAGATCAATGCCATCAAGGACACCCTGAACGAGACCATCAATAACAGCGAGGACGTGAACCACTTGTACATCAACTTCACCAGTCTCTCCTCCGGCGGCACCGCCTGGACCAGCCCGTACTACTACGCGAGTCGTATCAACCCCGAGATCGCCAACTACATCAAACAGAAAAACCCCACCCGGGTCGGTTGGATCATCCAGGACTTCATCAACGAGAAGTGGCACCCGCTGCTGTACCAGGAGGTGATCAACGCGAACAAATCGCTGGTGAAGTG ATGA

Data comparing 30 L fermentations of G2 (SEQ ID NO:8) and its codonoptimized versions (SEQ ID NO:9 and SEQ ID NO:10) is summarized in Table24. Fermentations were in P. fluorescence system described earlier. Datacomparing thermotolerance between codon optimized SEQ ID NO:10 vs. SEQID NO:8 is summarized in Table 25. Thermotolerance was measured asdescribed earlier.

TABLE 24 Time SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 24 1.90E+06 283.31E+06 29 8.98E+05 32 3.82E+06 33 1.62E+06 36 4.94E+06 37 2.47E+06 404.81E+06 41 3.82E+06 44 5.46E+06 45 5.28E+06 46 4.38E+06 48 5.26E+06 496.04E+06 50 9.02E+06 52 5.64E+06 53 5.27E+06 54 1.14E+07 56 1.19E+07 582.02E+07 60 6.22E+06 62 1.93E+07 66 1.88E+07 70 1.53E+07 75 1.22E+07

TABLE 25 SEQ ID NO: 8 SEQ ID NO: 10 Room Temp ° C. 100.00% 100.00% 55°C. 99.12% 134.05% 60° C. 116.64% 108.53% 65° C. 107.96% 108.16% 70° C.49.12% 56.78%

Data comparing activity of G2 (SEQ ID NO:8) and its codon optimizedversion (SEQ ID NO:10) is summarized in Table 26. The assays wereperformed using the Small scale oil procedure as described above.

TABLE 26 AVERAGED DATA Total 1- Sample # Experiment PA(%) PE(%) PI(%)PC(%) PL(%) LPA(%) LPE(%) LPI(%) LPC(%) LPA(%) 1 SEQ ID NO: 8—0.4 U/g0.18 0.48 0.02 0.47 1.14 0.00 0.02 0.00 0.03 0.00 2 SEQ ID NO: 8—0.2 U/g0.16 0.43 0.00 0.43 1.01 0.00 0.01 0.00 0.02 0.00 3 SEQ ID NO: 8—0.1 U/g0.18 0.46 0.05 0.50 1.19 0.01 0.01 0.00 0.03 0.00 4 SEQ ID NO: 8—0.05U/g 0.16 0.43 0.05 0.44 1.08 0.01 0.02 0.00 0.03 0.00 5 SEQ ID NO:10—0.4 U/g 0.19 0.51 0.00 0.53 1.23 0.01 0.02 0.00 0.03 0.00 6 SEQ IDNO: 10—0.2 U/g 0.15 0.41 0.00 0.44 1.00 0.01 0.02 0.00 0.03 0.00 7 SEQID NO: 10—0.1 U/g 0.15 0.41 0.07 0.41 1.05 0.00 0.02 0.00 0.02 0.00 8SEQ ID NO: 10—0.05 U/g 0.16 0.45 0.07 0.45 1.13 0.00 0.02 0.00 0.03 0.009 No Enzyme (Control) 0.17 0.46 0.23 0.47 1.33 0.00 0.02 0.02 0.03 0.001- 1- 1- Total P Total P from Sample # Experiment LPE(%) LPI(%) LPC(%)A(%) E(%) I(%) C(%) (ppm) PLs (ppm) 1 SEQ ID NO: 8—0.4 U/g 0.00 0.000.00 0.01 0.00 0.16 0.00 804.49 485.05 2 SEQ ID NO: 8—0.2 U/g 0.00 0.000.00 0.01 0.00 0.16 0.00 743.13 429.86 3 SEQ ID NO: 8—0.1 U/g 0.00 0.000.00 0.01 0.00 0.10 0.00 794.03 500.55 4 SEQ ID NO: 8—0.05 U/g 0.00 0.000.00 0.01 0.00 0.08 0.00 710.86 453.40 5 SEQ ID NO: 10—0.4 U/g 0.00 0.000.00 0.01 0.00 0.24 0.00 943.30 523.00 6 SEQ ID NO: 10—0.2 U/g 0.00 0.000.00 0.01 0.00 0.16 0.00 731.12 423.97 7 SEQ ID NO: 10—0.1 U/g 0.00 0.000.00 0.01 0.00 0.04 0.00 632.84 440.85 8 SEQ ID NO: 10—0.05 U/g 0.000.00 0.00 0.01 0.00 0.05 0.00 681.24 476.04 9 No Enzyme (Control) 0.000.00 0.00 0.01 0.00 0.00 0.00 711.84 551.23

Dosage

Dosage of enzyme may be defined by the number of “units” of enzyme to beadded per gram of oil to be treated, where one unit (U) is defined asthe quantity of enzyme required to liberate 1 umole of4-methylumbelliferone from 4.5 mM 4-Methylumbelliferylmyo-inositol-1-phosphate, N-methyl-morpholine salt in one minute at pH7.5 and 30° C. In one embodiment, dosage of PLC (e.g. using SEQ ID NO:2)or ePLC ranges from 1-50 U/g of oil, while dosage of PI_PLC ranges from0.05-20 U/g of oil. In another embodiment, dosages in the range of 1-20U/g of oil of PLC or ePLC and 0.05-2 U/g of oil of PI-PLC are used. Inanother embodiment, dosages in the range of 2-10 U/g of oil of PLC orePLC and 0.1-1 U/g of oil of PI-PLC are used. In an alternativeembodiment, dosage of PLC (e.g., using SEQ ID NO:2) or ePLC is 5.5 U/gof oil and for PI-PLC is 0.2 U/g of oil.

Alternatively, dosage may be determined by using the specific activityof the enzyme to convert the number of units of enzyme required per gramof oil to the weight of enzyme (ug) required per gram of oil.Specifically, the number of units required is divided by the specificactivity (U/mg) of the enzyme to arrive at the required ug of enzyme.For example, a dose of 0.2 U enzyme/gram of oil divided by a specificactivity of 90.1 U/mg, results in a dose of 2.22 ug of enzyme/g of oil.Therefore, in one embodiment, the dose of PI_PLC ranges from 0.55-222 ugof enzyme/g of oil. In another embodiment, dosages in the range of0.55-22.2 ug of enzyme/g of oil of PI-PLC are used. In anotherembodiment, dosages in the range of 1.11-11.1 ug of enzyme/g of oil ofPI-PLC are used. In an alternative embodiment, dosage of PI-PLC is 2.22ug of enzyme/g of oil.

In alternative embodiments, the invention also provides combinations ormixtures of enzymes comprising a PI-PLC of the invention and at leastone other enzyme, e.g., a phospholipase enzyme, e.g., a described inTable 8, or Table 9, or in WO 2008/036863. For example, in alternativeembodiments, the invention also provides combinations or mixtures ofenzymes comprising a PI-PLC of the invention and SEQ ID NO:2, not havinga signal sequence, encoded e.g., by SEQ ID NO:1; or SEQ ID NO:4, havinga signal sequence (equivalent to SEQ ID NO:2 with a signal sequence),encoded e.g., by SEQ ID NO:3; or including any of the ePLC enzymesdescribed in Example 2 (e.g., see Tables 8 and 9), and in WO2008/036863, which describe variants of SEQ ID NO:4 (encoded e.g. by SEQID NO:3). In alternative embodiments, the invention also providescombinations or mixtures of enzymes comprising a PI-PLC of theinvention, e.g., variants of SEQ ID NO:6 (encoded e.g. by SEQ ID NO:5)as described herein, or variants of SEQ ID NO:8 (encoded e.g. by SEQ IDNO:7, and the codon optimized SEQ ID NO:9 and SEQ ID NO:10).

Examples 4-9 are control examples. Examples 10-16 describe exemplarymethods provided herein.

In each of the examples below, the overhead mixer was IKA's RW 20digital with a flat blade paddle; operated at 200 rpm for normalagitation and 350 rpm for vigorous agitation. The centrifuge was a DeLaval Gyro—Tester installed with “The Bowl Unit” for continuousseparation. The centrifuge bowl was closed with the plug screwsinstalled. Shear mixing was accomplished with IKA's Ultra-Turraxhomogenizer T-50 basic with a S 50 N-G 45 G dispersion element at 10,000rpm. The PLA1 enzyme was Lecitase® Ultra (lot number LYN05015Acontaining 11.7 Units/mg) sold by Novozymes A/S of Denmark. The PLCenzyme was Purifine® PLC (lot number 90BU006A1 or 190DU001A1 containing26.0 or 28.6 Units/mg respectively) and the PI-PLC (SEQ ID NO:8)containing 4 Units/mg were provided by Verenium Corporation of SanDiego, Calif. The reaction took place in a 4 liter beaker and wascovered with a plastic wrap to reduce or eliminate any loss of water.The amount of phospholipids remaining in the treated oil was measured asppm P in accordance with the method of American Oil Chemists' SocietyOfficial Method Ca 20-99, “Analysis of Phosphorus in Oil by InductivelyCoupled Plasma Optical Emission Spectroscopy.” The Free Fatty Acid (FFA)was measured utilizing the American Oil Chemists' Society OfficialMethod Ca 5a-40. Moisture was measured using American Oil Chemists'Society Official Method Ca 2c-25. Neutral oil was measured using themethod set forth in the Appendix below. Acetone-insoluble materincluding phospholipids was measured using American Oil Chemists'Society Official Method Ja 4-46. Acid Value was measured using AmericanOil Chemists' Society Official Method Ja 6-55. The P-31 NMR proceduresand the Diacylglycerol (DAG) measurements by High Performance LiquidChromatography with Evaporative Light Scattering Detector (HPLC-ELSD),were performed by the procedures as reported by Verenium Corporation(then known as Diversa Corporation), “Analytical Profiling of SmallScale Reactions of phospholipase-C mediated Vegetable Oil Degumming,” atthe American Oil Chemists Society 2007 meeting.

Example 4 Control—Water Degumming at a Neutral pH

2001.2 grams of crude soybean oil containing 696.3 ppm of phosphorus washeated to 70-74° C. under normal agitation utilizing an overhead mixer.To the warm oil, 60.0 grams of de-ionized water was added. The oil/watermixture was agitated at 450 rpm for 1 minute. The agitator was slowed to100 rpm for 30 minutes to flocculate and hydrate the gums. The watertreated oil was then centrifuged; and the separated oil and wet gumswere collected. The residual phosphorus in the water degummed oil was80.0 ppm, FFA was 0.22%, and the DAG was 0.34%. The collected wet gumsweighed 108.5 grams.

Example 5 Control—PLC Degumming at a Neutral pH

2003.8 grams of crude soybean oil containing 696.3 ppm of phosphorus washeated to 60° C. under normal agitation utilizing an overhead mixer.With the temperature maintained at 60° C., 0.50 grams of Verenium'sPurifine® PLC lipase (lot number 90BU006A1) was added followed by 60grams of de-ionized water and the entire mixture was shear mixed for 60seconds. The oil mixture was agitated at normal speed for 120 minutes ata temperature of 60° C. The enzyme treated oil was then centrifuged; andthe separated oil and wet gums were collected. The residual phosphorusin the neutral pH PLC treated oil produced a degummed oil with aresidual phosphorus of 47.0 ppm. The FFA was 0.20% and DAG was 0.61%.The collected wet gums weighed 85.5 grams.

Example 6 Control—PI-PLC (SEQ ID NO:8) Degumming at a Neutral pH

2001.8 grams of crude soybean oil containing 696.3 ppm of phosphorus washeated to 60° C. under normal agitation utilizing an overhead mixer.With the temperature maintained at 60° C., 0.0110 grams of a PI-PLC (SEQID NO:8) provided by Verenium was added to a 10 ml beaker and dissolvedinto 1 ml of de-ionized water. Once the protein had dissolved in thewater, the enzyme solution was added to the oil. The beaker was rinsedthree times with approximately 1 ml of water in order to insure that allof the enzyme was added. The remainder of the water was added for atotal amount of water added was 60 grams. The oil enzyme water mixturewas shear mixed for 60 seconds. The oil mixture was agitated at normalspeed for 120 minutes at a temperature of 60° C. The enzyme treated oilwas then centrifuged; and the separated oil and wet gums were collected.The residual phosphorus in the neutral pH PI-PLC treated reactionproduced a degummed oil was 50.3 ppm. The FFA was found to be 0.18% andthe DAG was 0.49%. The collected wet gums weighed 102.5 grams.

Example 7 Control—PLC plus PI-PLC (SEQ ID NO:8) Degumming at a NeutralpH

2000.2 grams of crude soybean oil containing 696.3 ppm of phosphorus washeated to 60° C. under normal agitation utilizing an overhead mixer.With the temperature maintained at 60, 0.50 grams of Verenium'sPurifine® PLC lipase (lot number 90BU006A1) was added to the oil flowedby 0.0104 grams of a PI-PLC (SEQ ID NO:8) provided by Verenium was addedto a 10 ml beaker and dissolved into 1 ml of de-ionized water. Once theprotein had dissolved, the enzyme solution was added to the oil. Thebeaker was rinsed three times with approximately 1 ml of water in orderto insure that all of the enzyme was added to the oil. The remainder ofthe water was added for a total volume of water added was 60 grams. Theoil mixture was agitated at normal speed for 120 minutes at atemperature of 45° C. The enzyme treated oil was then centrifuged; andthe separated oil and wet gums were collected. The residual phosphorusin the PLA1-PI-PLC at neutral pH treated reaction produced degummed oilwith a residual phosphorus of 50.0 ppm. The FFA was 0.24% and DAG was0.85%. The collected wet gums weighed 84.2 grams.

Example 8 Control—PLA₁ Degumming at a Neutral pH

2000.6 grams of crude soybean oil containing 694.1 ppm of phosphorus washeated to 45° C. under normal agitation utilizing an overhead mixer.With the temperature maintained at 45° C., 0.10 grams of Novozymes'Lecitase® Ultra (PLA1 lipase lot number LYN05015) was added followed by60 grams of de-ionized water and the entire mixture was shear mixed for60 seconds. The oil mixture was agitated at normal speed for 240 minutesat a temperature of 45° C. The enzyme treated oil was then centrifuged;and the separated oil and wet gums were collected. The residualphosphorus in the PLA1 at neutral pH treated reaction produced adegummed oil with a residual phosphorus of 27.2 ppm. The FFA was 0.60%and DAG was 0.33%. The collected wet gums weighed 90.0 grams.

Example 9 Control—PLA₁ Degumming at pH 4.5

2001.2 grams of crude soybean oil containing 641.6 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool with agitation at normal speed untilthe oil temperature was 45° C., then 1.8 milliliters of 4 molar sodiumhydroxide solution was added to the oil and mixed. 0.10 grams ofNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05015) was addedfollowed by a total of 60 grams of de-ionized water and the entiremixture was shear mixed for 60 seconds. The oil mixture was agitated atnormal speed for 240 minutes at a temperature of 45° C. The enzymetreated oil was then centrifuged; and the separated oil and wet gumswere collected. The residual phosphorus in the PLA1 at a pH of 4.5treated oil produced a degummed oil with a residual phosphorus of 0.5ppm. The FFA was 0.54% and DAG was 0.33%. The collected wet gums weighed91.4 grams.

The degumming processes described in Examples 4-10 failed to removeand/or react all of the phospholipids present in the crude oil as isevident by the residual phosphorus, except for experiment 6. Experiment6 was depicts degumming at normal reaction conditions for Lecitase®Ultra enzyme. In Table 27, the phospholipid profiles of the collect wetgums are listed.

TABLE 27 Analytical results from the neutral control examples. ReactionWet Enzyme Water Temp Time Phos FFA DAG Gums Run Addition pH (g) (C.)(minutes) (ppm) (%) (%) (g) Starting Neutral — — — 668.0 0.53 0.32 —Material 1 None Neutral 60 70 30 80.0 0.22 0.34 108.5 2 PLC Neutral 6060 120 47.0 0.20 0.61 85.5 3 PI-PLC Neutral 60 60 120 50.3 0.18 0.49102.5 4 PLC + Neutral 60 60 120 50.0 0.24 0.85 84.2 PI-PLC 5 PLA1Neutral 60 45 240 27.2 0.60 0.33 90.0 6 PLA1 4.5 60 45 240 0.5 0.54 0.3391.4

As seen in Table 29, water degumming, demonstrates the emulsificationability of the hydratable phospholipids enabling the removal all speciesof phospholipids, even some of the non-hydratable species. However, alarge portion of the NHPs remained in the oil as evident by the residualphosphorus, calcium, magnesium and iron in the degummed oil (see Table29). Degumming utilizing a phospholipase C, was able to reactessentially all of the phosphatidylcholine and a significant amount ofthe phosphatidylethanolamine. Phosphatidylinositol and phosphatidic acidwere unreacted. The amount of collected gums, was significantly lowerthan the water degumming example (85.5 grams versus 108.5 grams), but alarge amount of the NHPs remained in the oil as evident by the residualphosphorus of near 50 ppm and the remaining Ca, Mg, and Fe left in theoil. As seen in Table 29, degumming with a phosphatidylinositol specificphospholipase, produced a wet gum with the highest amount of PC and PEof all of the control examples. Phospholipid composition of therecovered wet gums in the Examples 4-10 is also provided in FIG. 13.

TABLE 28 Phospholipid Composition of the recovered gums. PC PE PI PAlyso-PC lyso-PE Lyso-PI lyso-PA C E I A Run (%) (%) (%) (%) (%) (%) (%)(%) (%) (%) (%) (%) 1 9.62 7.35 4.02 2.04 0.58 0.35 0.33 b.d. b.d. b.d.b.d. 0.07 2 0.19 2.05 5.29 2.88 0.44 0.09 0.39 0.28 3.92 2.42 b.d. 0.193 11.01 7.78 0.00 2.33 0.59 0.36 b.d. b.d. 0.05 0.02 4.29 0.12 4 2.393.09 1.68 2.77 0.44 0.17 b.d. 0.35 3.25 1.97 1.26 0.18 5 0.24 0.41 0.670.26 8.05 7.33 4.32 3.52 b.d. b.d. b.d. 0.19 6 0.62 0.74 0.90 0.13 7.987.40 4.30 4.28 b.d. b.d. b.d. 0.25 b.d. = below detection limits

All of the PI was reacted with the phospholipase C producingphosphoinositol. Like examples 4 and 5, large amounts of NHPs remainedin the oil as is evident by the trace metal analysis reported in Table29.

TABLE 29 Elemental results from the neutral control examples. Phos-Magne- Enzyme phorus Calcium sium Iron Run Addition pH (ppm) (ppm) (ppm)(ppm) Average Starting Neutral 668.0 61.67 69.92 0.95 Crude Material 1None Neutral 80.0 34.97 18.79 0.44 2 PLC Neutral 47.0 21.25 10.14 0.19 3PI-PLC Neutral 50.3 25.33 12.33 0.20 4 PLC + Neutral 50.0 22.49 9.620.20 PI-PLC 5 PLA1 Neutral 27.2 15.14 7.73 0.13 6 PLA1 4.5 0.5 0.19 0.12b.d.

Moisture and neutral oil present in the recovered gums oil is reportedin Table 29a.

TABLE 29a Moisture and Neutral Oil present in the recovered gumsMoisture Oil “as is” Oil “dry” Oil Lost Run (%) (%) (%) (g) 1 43.3120.40 36.05 22.13 2 49.62 17.04 33.82 14.57 3 40.70 23.97 40.42 24.57 446.80 20.44 38.42 17.21 5 44.33 11.63 20.89 10.47 6 45.00 14.52 26.4013.27

In Example 7, degumming with both PLC and PI-PLC demonstratedsignificant enzymatic hydrolysis of PC, PE, and PI (Table 27 and FIG.13), but not as good as the single enzyme added alone in either Example5 or 6. Additionally, the residual phosphorus in the oil remained atapproximately 50 ppm due to the inability of the process to hydrolyzethe salts of phosphatidic acid and phosphatidylethanolamine. In Example8, degumming with PLA1 hydrolyzed almost all for the phospholipidspresent in the wet gums into their lyso-forms as seen in FIG. 13 and asis evident by the significant amount of lyso-phospholipids present(Table 28). However, even after 4 hours, significant amounts of PAremained in the oil because the NHPs were not accessible to the enzyme.

In the control experiment 6, PLA1 treated oil at pH 4.5 demonstrates theability of the phospholipase A1 to react with all of the phospholipidspecies producing a degummed oil with less than 1 ppm residualphosphorus and essentially all of the gums have been converted to theirwater soluble lyso-species as is evident in FIG. 13 and Tables 30 and31. The calcium, magnesium, and iron cations are no longer attached tothe NHPs, but are now in the water phase. The calcium, magnesium, andiron cations are dissociated from the salts of phosphatidic acid andphosphatidylethanolamine. As a consequence, the divalent elements maynow react with the citric acid present in the water phase forming acalcium, magnesium, or iron citrate that are insoluble in the waterphase at the reacted pH of the water phase. These insoluble saltsprecipitate out of solution forming a “hard water” coating on all of thepiping, heat exchangers, and even the centrifuge until the heavy phasecomprising mixture of reacted gums, water, and salts is removed via thecentrifugation process. Dayton et al. in U.S. 2007/0134777 disclose animproved enzymatic degumming process wherein the pH of the weaklybuffered enzymatic reaction is lowered from 4.5 to, for example, a rangeof 3.5 to 4.2 after the enzymatic reaction is completed dissociating thecitrate salts, thereby eliminating the fouling of the equipment,particularly the heat exchangers and the separating centrifuge, thatwould have resulted from precipitation of calcium and magnesium salts atthe optimum pH required for the enzyme activity.

As demonstrated by the following examples, the processes provided hereincomprise treating an oil with an acid to lower the pH to less thanthree, allowing the salts of phosphatidic acid and phosphatidylethanolamine (the NHPs) to dissociate, then adjusting the pH of thewater phase back to a neutral pH of 7, before hydrating thephospholipids in water degumming or reacting the phospholipids with aphospholipase allows the NHPs to migrate to the oil water interfaceallowing for better hydration and better substrate availability (NHPs)for the phospholipases. Table 30 below provides a summary of examples ofthe processes conducted as described below.

TABLE 30 Analytical results from the pH adjusted examples. Reaction WetEnzyme Adjusted Water Temp Time Phos FFA DAG Gums Run Addition pH (g)(C.) (minutes) (ppm) (%) (%) (g) 7 None 7.0 60 70 30 26.9 0.24 0.39112.0 8 PLC 7.0 60 60 120 14.4 0.21 1.11 80.0 9 PI-PLC 7.0 60 60 12013.5 0.22 0.61 100.8 10 PLC + 7.0 60 60 120 18.7 0.21 1.04 84.4 PI-PLC11 PLA1 5.5 60 45 240 1.6 0.54 0.33 91.7 12 PLA1 6.5 60 45 240 3.4 0.510.33 88.7 13 PLA1 7.0 60 45 240 3.4 0.52 0.39 93.1

Example 10 Water Degumming at an Adjusted pH of 7.0

2002.2 grams of crude soybean oil containing 640.1 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. 3.65 milliliters of 4 molar sodium hydroxide solution was addedto the oil and mixed. To the warm oil, 56.0 grams of de-ionized waterwas added (a total of 60 grams of water including the citric acid andsodium hydroxide). The oil/water mixture was agitated at 450 rpm for 1minute. The agitator was slowed to 100 rpm for 30 minutes to flocculateand hydrate the gums. The water treated oil was then centrifuged; andthe separated oil and wet gums were collected. The residual phosphorusin the water degummed oil was 26.9 ppm, FFA was 0.24%, and the DAG was0.39%. The collected wet gums weighed 112.0 grams.

The residual phosphorus in example 10 where the pH was adjusted to 7(26.9 ppm P) compared to the original neutral pH example (80.0 ppm P)demonstrated that more of the phospholipids were available for eitherhydration or mechanical entrapment. Additionally, the concentrations ofboth phosphatidic acid and phosphatidylethanolamine (NHPs) wereincreased in the recovered gums as seen in Table 31.

TABLE 31 Phospholipid composition of the recovered gums from examples10-16. PC PE PI PA lyso-PC lyso-PE Lyso-PI lyso-PA C E I A Run (%) (%)(%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 7 7.62 7.78 4.19 2.81 0.59 0.480.34 0.26 b.d. b.d. b.d. 0.18 8 0.30 0.88 6.15 3.84 0.67 0.22 0.51 0.673.91 2.79 0.06 0.31 9 8.29 8.31 b.d. 3.11 0.71 0.56 b.d. 0.48 0.42 0.204.63 0.23 10 1.12 1.74 0.74 3.34 0.36 0.09 b.d. 0.59 3.87 2.62 1.85 0.3011 0.40 0.51 0.42 0.18 7.78 7.06 4.40 3.83 b.d. b.d. b.d. 0.24 12 0.300.50 0.50 0.09 8.47 7.72 4.66 4.10 b.d. b.d. b.d. 0.24 13 0.14 0.43 0.07b.d. 7.65 7.31 3.97 3.26 0.06 b.d. 0.10 0.24 b.d. = below detectionlimits

Moisture and neutral oil present in the recovered gums from examples10-16 is reported in Table 31a.

TABLE 31a Moisture and Neutral Oil present in the recovered gums.Moisture Oil “as is” Oil “dry” Oil Lost Run (%) (%) (%) (g) 7 45.5317.44 32.02 19.53 8 51.58 13.23 27.32 10.53 9 43.19 18.29 32.20 18.44 1050.33 17.64 35.51 14.89 11 44.71 12.43 22.48 11.40 12 44.44 11.07 19.929.82 13 43.25 10.37 18.27 9.65

Example 11 PLC Degumming at an Adjusted pH of 7.0

2002.0 grams of crude soybean oil containing 640.1 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool to 60° C. where 3.65 milliliters of 4molar sodium hydroxide solution was added to the oil and mixed. With thetemperature maintained at 60° C., 0.50 grams of Verenium's Purifine® PLClipase (lot number 190DU001A1) was added followed by the remainder ofthe 60 grams of de-ionized water and the entire mixture was shear mixedfor 60 seconds. The oil mixture was agitated at normal speed for 120minutes at a temperature of 60° C. The enzyme treated oil was thencentrifuged; and the separated oil and wet gums were collected. Theresidual phosphorus in the acid/base pH 7.0 adjusted PLC treated oilproduced a degummed oil with a residual phosphorus of 14.4 ppm. The FFAwas 0.21% and DAG was 1.11%. The collected wet gums weighed 80.0 grams.

Results from this experiment demonstrate both the removal of thephospholipids from the oil and the phospholipase's ability to convertphospholipids into diacylglycerols or oil. The diacylglycerols wereincreased from the starting oil (0.31%) or 0.34% in the control waterdegumming experiment to 1 to 1.11% in the enzyme degumming experiment atan adjusted pH of 7.0. The residual phosphorus in the oil was reducedfrom 47.0 ppm in the control experiment to 14.4 ppm in the pH adjustedexperiment. The other trace metals were also significantly reduced asdemonstrated in Table 32.

TABLE 32 Elemental results from examples 10-16 Phos- Magne- EnzymeAdjusted phorus Calcium sium Iron Run Addition pH (ppm) (ppm) (ppm)(ppm) 7 None 7.0 26.9 7.80 2.71 0.35 8 PLC 7.0 14.4 4.70 0.96 0.34 9PI-PLC 7.0 13.5 5.30 1.99 b.d. 10 PLC + 7.0 18.7 4.60 2.14 b.d. PI-PLC11 PLA1 5.5 1.6 0.82 0.34 0.01 12 PLA1 6.5 3.4 2.56 0.95 b.d. 13 PLA17.0 3.4 2.75 1.03 b.d.

It is important to recognize that the collected gums were reduced to thelowest level in all of the experiments in the application (80.0 grams).The phospholipid composition shows that nearly all of the PC and PE wereconverted to the phospho-choline and phospho-ethanolamine (Table 31)forming the diacylglycerols found in the recovered oil. The amount ofphosphatidic acid was also increased in the collected gums furtherdemonstrating the increased availability of the substrate to either behydrated/mechanically “trapped” or available to be enzymaticallyreacted.

Example 12 PI-PLC Degumming at an Adjusted pH of 7.0

2000.0 grams of crude soybean oil containing 694.1 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool to 60° C. where 3.65 milliliters of 4molar sodium hydroxide solution was added to the oil and mixed. With thetemperature maintained at 60° C., 0.0108 grams of a PI-PLC (SEQ ID NO:8)provided by Verenium was added to a 10 ml beaker and dissolved into 1 mlof de-ionized water. Once the protein had dissolved in the water, theenzyme solution was added to the oil. The beaker was rinsed three timeswith approximately 1 ml of water in order to insure that all of theenzyme was added. The remainder of the water was added for a totalamount of water added was 60 grams. The entire mixture was shear mixedfor 60 seconds. The oil mixture was agitated at normal speed for 120minutes at a temperature of 60° C. The enzyme treated oil was thencentrifuged; and the separated oil and wet gums were collected. Theresidual phosphorus in the acid/base pH 7.0 adjusted PI-PLC treated oilproduced a degummed oil with a residual phosphorus of 13.5 ppm. The FFAwas 0.22% and DAG was 0.61%. The collected wet gums weighed 100.8 grams.

As in the two previous examples, the amount of PA present in thecollected gums was increased in the pH adjusted experiments versus thecontrol experiments. Additionally, the greatest amount of PC, and PEwere present in the collected gums, hence the 100.8 grams of wet gums.Since the phosphatidylinositol specific phospholipase predominately onlyreacts with PI, the increase in DAG was less than the phospholipasereaction in experiment 8 and the phospho-inositol was the predominantphospho-compound present in the collected gums (Table 31).

Example 13 PLC Plus PI-PLC Degumming at an Adjusted pH of 7.0

2003.8 grams of crude soybean oil containing 641.6 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool to 60° C. where 3.65 milliliters of 4molar sodium hydroxide solution was added to the oil and mixed. With thetemperature maintained at 60° C., 0.50 grams of Verenium's Purifine® PLClipase (lot number 190DU001A1) was added to the oil flowed by 0.0094grams of a PI-PLC (SEQ ID NO:8) provided by Verenium was added to a 10ml beaker and dissolved into 1 ml of de-ionized water. Once the proteinhad dissolved, the enzyme solution was added to the oil. The beaker wasrinsed three times with approximately 1 ml of water in order to insurethat all of the enzyme was added to the oil. The remainder of the waterwas added for a total volume of water added to 60 grams. The entiremixture was then shear mixed for 1 minute. The oil mixture was thenagitated at normal speed for 120 minutes at a temperature of 60° C. Theenzyme treated oil was then centrifuged; and the separated oil and wetgums were collected. The residual phosphorus in the PLC-PI-PLC at anadjusted pH of 7.0 treated oil produced degummed oil with a residualphosphorus of 18.7 ppm. The FFA was 0.21% and DAG was 1.04%. Thecollected wet gums weighed 84.4 grams.

The limited amounts of PC, PE and PI collected in the gums (Table 31);the large amount of phospho-choline, phospho-ethanolamine, andphospho-inositol in the gums (Table 31); and the high level ofdiacylglycerol present in the recovered oil clearly demonstrates theenzymatic reaction of both the PLC and the PI-PLC in the crude oil. ThePA recovered in the gums was also greater than the control combinationof enzymes found in Example 7.

Example 14 PLA₁ Degumming at an Adjusted pH of 5.5

2001.2 grams of crude soybean oil containing 641.6 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool with agitation at normal speed untilthe oil temperature was 45° C., then 2.55 milliliters of 4 molar sodiumhydroxide solution was added to the oil and mixed. 0.10 grams ofNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05015) was addedfollowed by a total of 60 grams of de-ionized water and the entiremixture was shear mixed for 60 seconds. The oil mixture was agitated atnormal speed for 240 minutes at a temperature of 45° C. The enzymetreated oil was then centrifuged; and the separated oil and wet gumswere collected. The residual phosphorus in the PLA1 at a pH of 5.5treated oil produced a degummed oil with a residual phosphorus of 1.6ppm. The FFA was 0.54% and DAG was 0.33%. The collected wet gums weighed91.7 grams.

Example 15 PLA₁ Degumming at an Adjusted pH of 6.5

2001.6 grams of crude soybean oil containing 641.6 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool with agitation at normal speed untilthe oil temperature was 45° C., then 3.31 milliliters of 4 molar sodiumhydroxide solution was added to the oil and mixed. 0.10 grams ofNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05015) was addedfollowed by a total of 60 grams of de-ionized water and the entiremixture was shear mixed for 60 seconds. The oil mixture was agitated atnormal speed for 240 minutes at a temperature of 45° C. The enzymetreated oil was then centrifuged; and the separated oil and wet gumswere collected. The residual phosphorus in the PLA1 at a pH of 6.5treated oil produced a degummed oil with a residual phosphorus of 3.4ppm. The FFA was 0.51% and DAG was 0.33%. The collected wet gums weighed88.7 grams.

Example 16 PLA₁ Degumming at an Adjusted pH of 7.0

2001.6 grams of crude soybean oil containing 641.6 ppm of phosphorus washeated to 70° C. under normal agitation utilizing an overhead mixer. 2.0grams of 50% w/w solution of citric acid was added and sheared for 1minute. The oil underwent normal agitation for 1 hour with an overheadmixer. The oil was allowed to cool with agitation at normal speed untilthe oil temperature was 45° C., then 3.31 milliliters of 4 molar sodiumhydroxide solution was added to the oil and mixed. 0.10 grams ofNovozymes' Lecitase® Ultra (PLA1 lipase lot number LYN05015) was addedfollowed by a total of 60 grams of de-ionized water and the entiremixture was shear mixed for 60 seconds. The oil mixture was agitated atnormal speed for 240 minutes at a temperature of 45° C. The enzymetreated oil was then centrifuged; and the separated oil and wet gumswere collected. The residual phosphorus in the PLA1 at a pH of 7.0treated oil produced a degummed oil with a residual phosphorus of 3.4ppm. The FFA was 0.52% and DAG was 0.39%. The collected wet gums weighed93.1 grams.

Examples 14, 15, and 16 demonstrate the effect of increasing the pH fromthe optimum condition for the enzyme of 4.5 to an adjusted pH from 5.5,6.5, and 7.0 respectively for the phospholipase A enzyme. The ability tooperate the reaction at a neutral pH of 7.0, instead of 4.5, wouldincrease the economical viability of the industrial process due tomaterials of construction and the cost of lowering the pH to 3.5 to 4.2in order to prevent the fouling of the industrial equipment. In summary,experiment 13 clearly shows that a low residual phosphorus in the oilmay be obtained, while still being able to react all of thephospholipids present in the gums to their lyso-forms thereforemaximizing the oil yield for this type of enzymatic reaction.

FIG. 14 compares the phospholipid compositions of the control neutral pHreactions versus the pH adjusted reactions at a pH of 7.0, side-by-side.It can be clearly seen that in all of the reactions where an acidtreatment followed by a dilute caustic treatment in order to bring thewater phase to a neutral pH increases the level of NHPs in the collectedgums and/or allows these NHPs to be converted to either lyso- orphospho-forms thereby increase the overall yield and minimizing anywaste products generated from the process.

FIG. 15 compares the neutral oil lost to the gum phase in the controlneutral pH reactions versus the pH adjusted reactions at a pH of 7.0,side-by-side. It can clearly be seen that in all of the reactions wherean acid treatment was followed by a dilute caustic treatment in order tobring the water phase to a neutral pH, the amount of neutral oil lost tothe gum phase decreased.

FIG. 16 compares the neutral oil lost to the gum phase of various pHconditions where a phospholipase A 1 is utilized. All of the reactionshave an adjusted pH except the “neutral” reaction. It is demonstratedthat the amount of neutral oil is lowest when the pH has been adjustedto 7.0 versus even the neutral unadjusted reaction without any acid orbase added to aid in the availability of the NHP to the degummingprocess.

Example 17 Particle Size Distribution Studies

In this example, droplet size average distribution and prevalent dropletsize of the shear treatment applied in the methods provided herein andmethods available in the art (for example, U.S. Pat. Nos. 4,698,185 and6,103,505) were studied.

The particle size in this study was analyzed using the MalvernMastersizer particle (droplet) size analyzer model 2000, with particleand volume distribution calculated by the software that accompanies theequipment.

Example 17A

300 g of degummed soybean oil was heated to 90° C. in a 600 ml beakerunder normal agitation utilizing a magnetic stirrer. To this was added1.8 g of deionized water, followed by 0.565 g of phosphoric acid 85%.The mixture was then sheared for 30 seconds using a Ultra-Turraxhomogenizer T-50 basic with a S 50 N-G 45 G dispersion element at 10,000(this homogenizer is believed by the supplier to be equivalent to theUltra Turrax T-45 used in U.S. Pat. Nos. 4,698,185).

A sample of the emulsion formed was then analyzed in the Malvern 2000.The average droplet size for the aqueous phase was 3.963 μm and 90% ofthe aqueous phase appeared in droplets with diameter less than 6.675 μm(Table 33). The droplet distribution is shown in FIG. 17.

TABLE 33 volume distribution (%) accumulated volume distribution (%)Droplet Example Example Example Example Example Example Example ExampleSize (μm) 17A 17B 17D 17C 17A 17B 17D 17C 1.259 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 1.445 0.13 0.00 0.01 0.00 0.13 0.00 0.01 0.00 1.660 2.380.91 0.09 0.01 2.51 0.91 0.10 0.01 1.905 5.25 4.62 0.10 0.10 7.76 5.530.20 0.11 2.188 7.63 7.51 0.09 0.23 15.39 13.04 0.29 0.34 2.512 9.379.76 0.08 0.35 24.76 22.80 0.37 0.69 2.884 10.44 11.18 0.15 0.49 35.2033.98 0.52 1.18 3.311 10.84 11.71 0.37 0.67 46.04 45.69 0.89 1.85 3.80210.62 11.45 0.87 0.90 56.66 57.14 1.76 2.75 4.365 9.95 10.60 1.73 1.2066.61 67.74 3.49 3.95 5.012 8.95 9.35 2.98 1.60 75.56 77.09 6.47 5.555.754 7.72 7.89 4.62 2.12 83.28 84.98 11.09 7.67 6.607 6.30 6.27 6.502.77 89.58 91.25 17.59 10.44 7.586 4.78 4.58 8.49 3.56 94.36 95.83 26.0814.00 8.710 3.21 2.92 10.26 4.46 97.57 98.75 36.34 18.46 10.000 1.801.23 11.51 5.46 99.37 99.98 47.85 23.92 11.482 0.62 0.00 11.94 6.4599.99 100.00 59.79 30.37 13.183 0.01 0.00 11.41 7.30 100.00 100.00 71.2037.67 15.136 0.00 0.00 9.99 7.97 100.00 100.00 81.19 45.64 17.378 0.000.00 7.90 8.27 100.00 100.00 89.09 53.91 19.953 0.00 0.00 5.57 8.23100.00 100.00 94.66 62.14 22.909 0.00 0.00 3.34 7.81 100.00 100.00 98.0069.95 26.303 0.00 0.00 1.61 7.06 100.00 100.00 99.61 77.01 30.200 0.000.00 0.39 6.08 100.00 100.00 100.00 83.09 34.674 0.00 0.00 0.00 4.97100.00 100.00 100.00 88.06 39.811 0.00 0.00 0.00 3.85 100.00 100.00100.00 91.91 45.709 0.00 0.00 0.00 2.82 100.00 100.00 100.00 94.7352.481 0.00 0.00 0.00 1.96 100.00 100.00 100.00 96.69 60.256 0.00 0.000.00 1.28 100.00 100.00 100.00 97.97 69.183 0.00 0.00 0.00 0.79 100.00100.00 100.00 98.76 79.433 0.00 0.00 0.00 0.47 100.00 100.00 100.0099.23 91.201 0.00 0.00 0.00 0.27 100.00 100.00 100.00 99.50 104.713 0.000.00 0.00 0.17 100.00 100.00 100.00 99.67 120.226 0.00 0.00 0.00 0.11100.00 100.00 100.00 99.78

Example 17B

2000 g of degummed soybean oil was heated to 90° C. in a 4000 ml beakerunder normal agitation utilizing a magnetic stirrer. 12 g of deionizedwater was added to the oil, followed by 3.767 g of phosphoric acid 85%.The mixture was then sheared for 30 seconds using a Ultra-Turraxhomogenizer T-50 basic with a S 50 N-G 45 G dispersion element at10,000.

A sample of the emulsion formed was then analyzed in the Malvern 2000.The average droplet size for the aqueous phase was 3.905 μm and 90% ofthe aqueous phase appeared in droplets with diameter less than 6.405 μm(Table 33). The droplet distribution is shown in FIG. 18.

Example 17C

2000.3 g of crude soybean oil was heated to 60° C. in a 4000 ml beakerunder normal agitation utilizing a magnetic stirrer. 2.0 grams of 50%w/w solution of citric acid was added and sheared for 1 minute. 3.650milliliters of 4 molar sodium hydroxide solution was added to the oiland mixed. 0.50 grams of Purifine PLC enzyme was added followed by atotal of 60 grams of de-ionized water and the entire mixture was shearmixed for 30 seconds using a Ultra-Turrax homogenizer T-50 basic with aS 50 N-G 45 G dispersion element at 10,000 rpm.

A sample of the emulsion formed was then analyzed in the Malvern 2000.The average droplet size for the aqueous phase was 19.957 μm and 90% ofthe aqueous phase appeared in droplets with diameter less than 36.998 μm(Table 33). The droplet distribution is shown in FIG. 19.

Example 17D

0.6 L (approximately 560 g) of degummed soybean oil was heated to 40° C.in a 600 ml beaker under recirculation through the Silverson mixer. Themixer was cooled in a water bath, to avoid temperature increase duringthe experiment. 0.6 g of 50% w/w solution of citric acid was added,followed by 1.095 milliliters of 4 molar sodium hydroxide solution.0.028 g (50 ppm) of Lecitase Ultra PLA enzyme was added followed by atotal of 16.8 g of de-ionized water.

A sample of the emulsion formed was then collected after 15 min, inorder to allow for the system equilibrium. The sample was then analyzedin the Malvern 2000. The average droplet size for the aqueous phase was12.691 μm and 90% of the aqueous phase appeared in droplets withdiameter less than 20.333 μm (Table 33). The droplet distribution isshown in FIG. 20. Being an in-line mixer, the Silverson continued toreduce droplet sizes, eventually achieving average particle size of 9μm.

FIG. 21 depicts superimposition of the droplet (particle) sizedistribution resulting from Examples 17A, 17B, 17C, and 17B.

Table 34 provides data for comparative particle size for Examples 17A,17B, 17C and 17D.

TABLE 34 Example Example Example Example 17A 17B 17D 17C Averageparticle size 3.963 3.905 12.691 19.975 (μm) particle size for 6.6756.405 20.333 36.998 accumulated water volume of 90% (μm) water phase indroplets 99.37 99.98 47.85 23.92 <=10 μm (%) water phase in droplets0.63 0.02 52.15 76.08 >10 μm (%)

As seen from the data, Example 17C produced a very distinctive dropletdistribution profile, with higher average particle size. The averageparticle size of Example 17C was found to be approximately 5 timesbigger than Example 17A and 17B, and 1.6 times bigger than Example 17D.

As seen from the data, for Example 17C, more than 76% of the water phaseis found in droplets with diameter bigger than 10 μm, while for Example17A 52% of the water phase is found in droplets with diameter biggerthan 10 μm, and less than 0.65% of the water phase is found in dropletswith diameter bigger than 10 μm in Example 17D.

Example 18 Comparative Study of Gums Obtained by Various Methods

In this example, comparative data is provided for oil treated accordingto processes reported in U.S. Pat. No. 4,698,185 and process describedin Examples 4-16 above.

Various oils (listed in Table 35) are treated according to the followingprotocol as described in U.S. Pat. No. 4,698,185:

-   -   oil heated to greater than 75° C.    -   water added to raise concentration to 0.6% wt    -   about 20-60 wt % phosphoric acid added into water degummed oil    -   acid dispersed for 30 seconds    -   mixing continued for three minutes    -   caustic solution added in about 2% by volume    -   the mixture centrifuged for 30 minutes at 5,000 rpm    -   the oil phase separated and washed with 2 wt % demineralized        water    -   washing water removed by centrifugation for 30 minutes at 5,000        rpm    -   gums in the oil analyzed

Table 35 below provides data for composition of gums separated from thetreated oil samples:

Corn Oil Type Soybean Sunflower Germ Rapeseed Groundnut Phospholipids PA55 49 37 72 53 LPA 6 20 — 6 — PC 4 8 26 <1 15 LPC <1 — — PE 17 9 13 13 7 LPE <1 5 2 Cardiolipin N- 13 9 19 7 24 acylphosphatidyl EthanolaminePI 4 — 5 — Not Identified — — —

Table 36 provides below provides data for phospholipids in oil samplestreated according to Examples 10, 11, 12, 13, and 16 above:

TABLE 36 Phospho- Example Example Example Example Example lipids 10 1112 13 16 PA 11.6 18.9 11.6 20.1 0.0 LPA 1.1 3.3 1.8 3.6 14.1 “A” 0.7 1.50.9 1.8 1.0 PC 31.4 1.5 31.0 6.7 0.6 LPC 2.4 3.3 2.7 2.2 32.9 “C” b.d.19.3 1.6 23.3 0.3 PE 32.1 4.3 30.4 10.5 1.9 LPE 2.0 1.1 1.0 0.5 31.5 “E”b.d. 13.7 11.2 15.8 b.d. PI 17.3 30.3 9.6 4.4 0.3 LPI 1.4 2.5 b.d. b.d.17.1 “I” b.d. 0.3 17.3 11.2 0.4

Table 37 below provides summary of enzymes used, pH of the reaction anddata for phospholipids in oil samples treated according to Examples 4,5, 6, 7, 8, 10, 11, 12, 13 and 16 above.

TABLE 37 PC PE PI PA LPC LPE LPI LPA C E I A Enzyme pH 1 39.5 30.2 16.58.4 2.4 1.4 1.4 0.0 0.0 0.0 0.0 0.3 None Neutral 7 31.4 32.1 17.3 11.62.4 2.0 1.4 1.1 0.0 0.0 0.0 0.7 None 7.0 2 1.1 11.3 29.1 15.9 2.4 0.52.2 1.6 21.6 13.3 0.0 1.1 PLC Neutral 8 1.5 4.3 30.3 18.9 3.3 1.1 2.53.3 19.3 13.7 0.3 1.5 PLC 7.0 3 41.5 29.3 0.0 8.8 2.2 1.3 0.0 0.0 0.20.1 16.2 0.5 PI-PLC Neutral 9 31.0 30.4 0.0 11.6 2.7 2.1 0.0 1.8 1.6 0.717.3 0.9 PI-PLC 7.0 4 13.6 17.6 9.6 15.8 2.5 1.0 0.0 2.0 18.5 11.2 7.21.0 PLC + Neutral PI-PLC 10 6.7 10.5 4.4 20.1 2.2 0.5 0.0 3.6 23.3 15.811.2 1.8 PLC + 7.0 PI-PLC 5 0.9 1.6 2.7 1.1 32.2 29.3 17.3 14.1 0.0 0.00.0 0.7 PLA1 Neutral 13 0.6 1.9 0.3 0.0 32.9 31.5 17.1 14.1 0.3 0.0 0.41.0 PLA1 7.0

As seen from the data, phospholipids obtained by the processes describedherein have a unique composition.

The embodiments of the claimed subject matter described above areintended to be merely exemplary, and those skilled in the art willrecognize, or will be able to ascertain using no more than routineexperimentation, numerous equivalents of specific compounds, materials,and procedures. All such equivalents are considered to be within thescope of the claimed subject matter and are encompassed by the appendedclaims.

While the invention has been described in detail with reference tocertain exemplary aspects thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

What is claimed is:
 1. A method for degumming a crude oil comprising:i-a) mixing an aqueous acid with the crude oil to obtain an acidicmixture having pH of less than about 4; i-b) mixing a base with theacidic mixture to obtain a reacted mixture having pH of about 6-9,wherein the mixing in steps i-a) and/or i-b) creates an emulsion thatcomprises at least about 60% of an aqueous phase by volume in dropletsize between about 15 μm to about 45 μm in size; and i-c) mixing aphospholipase enzyme with the emulsion in step (i-b), wherein thephospholipase enzyme comprises SEQ ID NO:8.
 2. The method of claim 1,wherein the emulsion comprises at least about 90% of the aqueous phaseby volume in droplet size of about 15-40 μm in size.
 3. The method ofclaim 1, wherein the acid is selected from the group consisting ofphosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid,and a mixture thereof.
 4. The method of claim 1, wherein the pH of theacidic mixture in step i-a) is about 1 to about
 4. 5. A method fordegumming a crude oil comprising: i-a) mixing an aqueous acid with thecrude oil to obtain an acidic mixture having pH of less than about 4;i-b) mixing a base with the acidic mixture to obtain a reacted mixturehaving pH of about 6-9, wherein the mixing in steps i-a) and/or i-b)creates an emulsion that comprises an aqueous phase in average dropletsize between about 15-35 μm; and i-c) mixing a phospholipase enzyme withthe emulsion in step (i-b), wherein the phospholipase enzyme comprisesSEQ ID NO:8.
 6. The method of claim 1, wherein the enzyme is apolypeptide having SEQ ID NO:
 8. 7. The method of claim 5, wherein theenzyme is a polypeptide having SEQ ID NO: 8.